Method for stimulating production of variable region gene family restricted antibodies through B-cell superantigen vaccination

ABSTRACT

Criteria for identifying potential B cell superantigens are disclosed, together with a method for determining whether these candidate antigens have B cell superantigenic activity. Methods for constructing and using a vaccine including B cell superantigens are also disclosed. Identification is based on characterizing the structure of Ig binding sites which interact with the candidate antigen assessment of Ig V region diversity on binding of candidate and conventional antigens, confirmation of sAg activity in interactions between candidate antigens and whole cells, confirmation of whether the candidate antigen induces B cell mitogenesis, determination of the earliest point in B cell development where cellular co-factors are required for sAg activity and, for reference, determination of V region usage in responder populations. Once a B cell superantigen is characterized, it is purified and conjugated by chemical means to a polysaccharide or glycoprotein component from a microbial capsule, cell wall, envelope or other component preferably using components which stimulate production of antibodies with the same V region restriction as antibodies whose production is stimulated by the B cell superantigen.

RELATED U.S. PATENT APPLICATIONS

This is a continuation-in-part of U.S. application Ser. No. 07/969,936,filed Oct. 30, 1992, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of Invention

The field of this invention relates to antigenic stimulation of specificimmune responses. Specifically, the invention relates to identificationof B cell superantigens and their use as adjuvants and/or carrierproteins to enhance a specific immune response to bacterial or viralpathogens having polysaccharide or glycoprotein components in their cellwalls, cell membranes, capsules or envelopes. More particularly, itrelates to a method of enhancing the immune response by administrationof said polysaccharide or glycoprotein with a B cell superantigen eitherconcomitantly or in a chemically conjugated form.

2. History of the Prior Art

For a complete understanding of the invention, a brief summary of therole of clonal development of B cells in immune responses is helpful.

In the germ-line cells, there are three sets of germ-line genes involvedin immunoglobulin coding: one set codes for the heavy chains, and theother two code for two types of light chain designated by the Greekletters kappa (κ) and lambda (λ), which differ significantly in theamino acid sequence of their constant domains. Immunoglobulins arecomposed of heavy and light chain heterodimers, which contribute to theconventional antigen binding site in the Fab portion. B cells themselvesare lymphocytes which have immunoglobins on their cell surface whichserve as receptors for antigens. Binding of antigen, in most instancestogether with additional signals from T cells, causes the original Bcell to proliferate, forming clones. The expanded clonal populationdifferentiates into memory cells and plasma cells, the latter of whichsynthesize and secrete antibodies which generally will have identicalbinding sites for the antigen which triggered the B cell activation.Polyclonal activation occurs when certain antigens (known to includemitogens and the Epstein-Barr virus) stimulate clonal expansionregardless of the antigen specificity of the B cells involved.

The human antibody repertoire is responsible for the acquisition of adynamic and responsive immune defense system, but dysregulation withinthe B cell compartment can result in a wide spectrum of clinicalconditions, including inappropriate B cell clonal expansions (e.g., Bcell neoplasms), inadequate immune defense from infection, or autoimmunedisease. During fetal development, antibody specificities are acquiredslowly in a developmentally ordered fashion.

Referring for purposes of illustration to humans, the V, J, and D genescode in human cells for the variable regions of the antibody moleculeand the C genes code for the constant regions. Each of the threegerm-line sets contain from two to at least 300 alternative V genes,together with a small number of alternative J genes; the heavy-chain setalso has alternative D genes. Any of these genes can contribute in thevariable regions of antibody molecules. Each set has from one to fivealternative C genes coding for different constant regions.

As a germ-line cell differentiates into a mature but "naive" B cell(i.e. one that is reactive to but has not yet encountered its matchingantigen), somatic recombination of the germ-line genes takes place. Ineach cell, one of the V genes from each of the three germ-line sets is"selected" by an unknovin molecular process, together with one of theadjacent J genes (and in heavy chains, also with one of the D genes).These selected genes are brought together in the genome when theintervening DNA is excised. The basis of diversity in theantigen-recognition structure of an antibody molecule rests initially onthis recombination event, since different genes are recombined indifferent B cells.

Although the first source of variation in the structure of the antibodymolecule is brought about by somatic recombination of alternative V, Dand J genes, further diversity in the amino acid sequence of thevariable domains results from variable recombinations; i.e., slightvariations in the exact location of the "cutting points" as first thegerm-line DNA and later the mRNA transcripts are cut and spliced. Boththese sources of variation occur before contact with antigen.

More variation arises after contact with antigen. Single base changes("somatic mutation") occur in the DNA of activated B cells, mainlyduring the process of memory-cell formation.

A consequence of somatic mutation is that some of the binding sitesproduced by the mutated DNA have a better affinity for the antigen, andsome have a worse affinity. Somatic mutations occur mainly during clonalexpansion and memory-B cell formation, so the memory B cells from asingle clone end up with receptors (i.e. surface immunoglobulin) for thesame antigen, but with a range of antigen affinities (see, redevelopment of the human antibody repertoire generally, Davey,Immunology: A Foundation Text (1990) Chapter 4, sections 4.3-4.3.2).

The process of repertoire selection may be the result of long-termexposure to many exogenous and endogenous contentional ligands, but adramatic skewing of the immune repertoire can also be induced by asingle limited exposure to certain unconventional antigens. Theseantigens, of bacterial or viral origin, were first distinguished basedon an ability to interact with a large proportion of T lymphocytes. Incontrast to conventional antigens, which generally stimulate less than0.01% of T cells, these superantigens can stimulate 5-25% of all Tcells. In explanation, many superarntgens are recognized by most (orall) T cells that use a particular Vβ family. Based on available data,superantigen reactivity is little affected by differences in Vβjunctional sequences, or by the alpha chains that are co-expressed.Moreover, recent studies indicate that an alternative site, remote fromconventional antigen binding sites, allows for superantigen recognitionby a large proportion of the T cell pool, usually those havingparticular Vβ elements.

Superantigens have been proposed to contribute to the shaping of themature T cell repertoire by clonal selection and/or deletion. Individualinbred strains of mice have been shown to carry different murine MammaryTumor Virus encoded endogenous superantigens, and each exhibits adistortion of the distribution of T cell receptor Vβ expression. This isusually due to deletion of T cells with certain Vβ families, althoughthere may also be more subtle effects on positive selection. Similaracute fluctuations may also occur in patients with toxic shock syndromeand Kawasaki's disease, presumably due to a T cell superantigen. Severalrecent papers have also suggested that superantigen exposure ofpredisposed individuals may, at times, result in immunosuppression, theproduction of autoantibodies, the development of autoimmune disease, orthe abolition of an autoimmune process (see also, re characteristics ofT cell superantigens, Fraser, et al. (1992) J. Exp. Med 175:1131-1134,and Taub, (1992) Cell Immunol. 140:267-281).

With this background, interest in the possibility that B cellsuperantigens (hereafter sAg) may exist can be understood. Severalcandidate sAg's are suggested by recent reports. For example, human IgM,IgA and IgGF(ab')₂ that bind to bacterial membrane proteinstaphylococcal protein A (SpA) have been shown to include a family ofV_(H) genes which encode polypeptides belonging to the V_(H) 3 proteinsubgroup (the largest human family) (Sasso, et al., J. Immunol. (1991)147:1877-1883 and Sasso, et al., J. Immunol. (1989) 142:2778-2783). Asdescribed further herein, SpA binds to the Fab region of a largeproportion of V_(H) 3 restricted immunoglobins at an alternative bindingsite different from its known, F_(C) γ binding sites and in greaterproportion than conventional antibody binding. Further, the Fab siteswhich bind SpA are found on antibodies with diverse specificities.

Protein F_(V), a sialoprotein released into the digestive tract duringviral infection, has also been reported to interact with the Fabfragment of immunoglobins at an unconventional binding site believed tobe in the V_(H) domain (Bouvet, Scand. J. Immunol. (1991) 33:381-386).Further, based on reported binding by human Ig via the Fab region (in asetting in which prior immunization to create antibodies to conventionalcomponents is not required), known protein components of othermicroorganisms may also serve as B cell sAg.

The present invention includes a means of characterizing and identifyingB cell sAg and using them to enhance production of V_(H), particularlyV_(H) 3, restricted antibodies. In particular, the sAg will beidentified, purified and administered concomitantly with apolysaccharide or glycoprotein component from a bacterial or viral cellwall or capsule or, preferably, as a carrier for a conjugate vaccine tothe bacteria or virus. B cell superantigens with specificity for otherVariable (V) region gene families may also exist, and be useful incertain vaccines.

In the past, there has been no rationale for selection of carriers forconjugate vaccines except the experience with these proteins asimmunogens themselves. In each case, diphtheria toxoid, tetanus toxoidand OMB (outer membrane protein of N. meningitides) the carriers wereselected and/or used clinically to elicit immunity to the pathogen. As aresult they were shown to be immunogenic and safe for human use.Prototype conjugate vaccines were then made and tested in animal models,usually rabbit or mice. The only possible exception is the present OMBconjugate, which uses a liposome delivery system (Donnely et al., J.lmmunol (1990) 145: 3071-3079).

SUMMARY OF THE INVENTION

In this application, the carrier or adjuvant sAg will be selected tospecifically enhance a patient's antibody repertoire by specificallycausing B cell activation and clonal expansion, in particular that whichwill produce V_(H) and V_(H) 3 restricted antibodies. This method isexpected to be particularly effective in assisting patients to mount adefense to invasive infection by encapsulated bacteria and virusesbecause that defense is highly dependent on the production of antibodiesspecific for capsular or cell wall polysaccharides or envelopeglycoproteins. Further, the very young and elderly members of apopulation, as well as certain ethnic populations of humans, are atincreased risk of these infections due to impairments in the productionof these antibodies. Use of a V region specific polyclonal activatorshould, therefore, be of particular value in treatment of theseindividuals.

Further, particularly when using a purified sAg, the potential fortoxicity is far lower than that reported for conventional conjugatevaccines, because the stimulatory activity of the sAg will be limited tothe B cell compartment. Compare this activity to, for example, theanti-haemophilus influenzae B (Hib) vaccine reported by Donnely, sugra,which uses an OMB protein carrier naturally contaminated with anendotoxin responsible for fever and other toxicity.

In addition, a B cell superantigen will by definition bind a largerproportion of Ig than would be bound by a conventional antigen. Thepercentage of surface Ig in a given B cell population which willnormally bind to a particular superantigen can be quantified. The B cellsuperantigen may therefore serve as a phenotypic marker for a B cellpopulation. Further, observed abnormalities in the binding pattern for aparticular superantigen in a given B cell population may signal theonset or existence of a malignancy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 diagrammatically depicts the pCOMB3 vector in its surface displayand Fab producing forms.

FIG. 2 diagrammatically illustrates the compositon of the pCOMB3 vectorand the proposed pathway for Fab assembly and incorporation into thefilamentous phage coat.

FIG. 3 is a reproduction of nitrocellulose fitters on whichantigen-specific clones are identified by autoradiography aftersuccessive rounds of panning with hyperiodinated SpA.

FIG. 4A through R is a reproduction of stained flow cytometry studiesshowing binding to normal human tonsillar mononuclear cells by ProteinG, SpA and hyperiodinated SpA. Protein G, SpA and hyperiodinated (mod)SpA are identified along the horizontal axis of each reproduction, whilethe antibody isotype (IgM or IgG) is identified along the vertical axisof each reproduction.

FIGS. 5-7 depict, in tabular form, the results of reactivity testsbetween human anti-polysaccharide bacterial antibodies and serologicreagents for variable region restriction of antibodies.

FIG. 8, A-C, depicts the results of an EUSA for the antibody response tomod-SpA immunization in Balb/c mice.

FIG. 9 depicts the results of an EUSA for inhibition of mouse Ig bindingto mod-SpA by human V_(H) 3 IgG Fab Sp A3-08.

FIG. 10 depicts the results of EUSA's for the antibody responses topneumococcal polysaccharide in mice immunized either with polysaccharidealone or polysaccharide and unconjugated mod-SpA. The terms Pre and Postin the FIGURE refer to antibody responses obtained before and afterantigen priming.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

A. CHARACTERISTICS OF sAg AND CANDIDATE ANTIGENS.

The first task in developing the inventive vaccine is to identify usefulB cell sAg. Based on known qualities of T cell superantigens (describedabove) and the analytic work disclosed below, it is proposed that B cellsuperantigens will possess the following structural and functionalproperties:

1. Structural properties.

A) Superantigens should interact with isolated Ig and surface Ig.

B) Superantigen binding should be expressed by a large proportion ofnon-immune Ig of different isotopes (constant regions). This proportionshould be much greater than might be expected to bind to a conventionalantigen (e.g.,>1%).

C) Superantigen binding should be present on antibodies that alsoexpress diverse conventional antigen binding activities.

D) Superantigen binding should be restricted by subgroup or V family,and this interaction should correlate with conserved sequences that areoutside of the conventional antigen binding site.

2. Functional properties.

A) In vitro stimulation with a superantigen should contribute to theselective stimulation of V_(H) family restricted B cell populations.

B) In vivo stimulation with superantigen should result in dramaticshifts in the presentation within the B cell repertoire, whichnumerically are much greater than can occur after hyperimmunization witha conventional antigen.

To characterize a candidate antigen as a B cell superantigen, analyticalmethods are described below which will confirm whether the antigenpossesses the above-listed characteristics. Depending on whether thecandidate antigen is already known to possess certain characteristics,certain steps of the approach described below may be viewed by thoseskilled in the art as optional. It will also be clear to those skilledin the art upon review of the disclosed approach that certain steps maybe accomplished by other means (using, for example, a different vectorin the development of combinatorial Fab libraries, or an analyticalmethod other than flow cytometry to gauge sAg candidate bindingspecificities). However, the methods identified below have proved to beefficient and useful means of characterizing a B cell sAg.

For purposes of clarity, it should be noted that the below approach hasbeen described in a specific context; i.e., toward characterizingstaphylococcal protein A (SpA) as a B cell sAg. However, the approach isnot so limited and can be applied to other candidate antigens as well.In this regard, candidate antigens for characterization as sAg based ontheir demonstrated binding to human Ig via the Fab region include:

1. Protein A (from Staphylcoccus aureus cell membrane!).

2. Protein L (from Peptococcus magnus in cell wall!).

3. Protein P (from Clostridia pertingens, cell surface component!) withmolecular weight (MW) of 190,000.

4. Protein B (from Group B Streptococcus certain strains! protein withMW of 25,000).

5. Brucella abortus (protein component from certain strains).

6. Taylorella equigeuitalis (whole bacteria)

7. Streptococcus zooepidemicus (whole bacteria).

8. Aeromonas salmonicida (protein component with MW of 49,000).

9. Protein F_(V) (human liver protein with V_(H) restricted binding toFab).

B. ANALYTIC METHODS FOR IDENTIFICATION OF sAg (USING SpA EXAMPLE).

Step 1. Structurally characterize the site on Ig receptors responsiblefor interaction with the candidate antigen.

i. Immunoblotting analysis.

Incorporated by reference herein is a disclosure of serologic reagentsto distinguish the products of V kappa gene families in humans. Each ofthese antisera identifies a sequence selected from a first framework(FW) region portion that is highly conserved by members of a particularV_(k) family, but differs from the homologous FW sequences from otherV_(k) families (Silverman, et al., (1986)) J. Immunol. Methods95:249-257). A second reference, also incorporated herein, disclosesreagents to discriminate the genetic origin of heavy (H) chains, basedon analyses of V region DNA sequence homology identifying at least sixdistinct V_(H) gene families (Berman, et al., (1988) EMBO, J. 7:727-738re V_(H) gene families!; see also (incorporated herein) Silverman, etal. (1988) J. Clin. Invest. 82:469-475; Silverman, et al. (1988) J. Exp.Med. 168:2361-2366; and, Silverman, et al. (1990) Arthritis Rheum.33:1347-1360 re reagents!).

Polyclonal or monoclonal Ig may be separated into binding andnon-binding fractions by, for example, passage over affinity columnssuch as agarose (e.g., "SEPHAROSE" having the candidate antigen bound tothe solid phase. These fractions are then analyzed by immunoblottingwith the V family specific serologic reagents (for further referencesee, incorporated herein, Sasso, et al., (1988) J. Immunol.140:3098-3107 re polyclonal and monoclonal IgM rheumatoid factorspecificities!; and, Sasso, et al. (1989) J. Immunol. 142:2778-2783 rehuman IgM specificities for SpA!).

ii. Application to SpA.

To apply this method to SpA, the binding activity of its purifiedrecombinant form (available from Calbiochem, La Jolla, Calif. under thetrademark "ULTRAPURE") is tested against panels of purified monoclonalIgM and IgG proteins that are representative of all known V_(k) andV_(H) families, as well as the binding activity of SpA modified byhyperiodination to destroy IgG Fc binding activity (mod-SpA). (It shouldbe noted that although as further noted herein other forms of SpA may beused, the recombinant form is preferred. Therefore, unless otherwiseindicated, SpA as referred to herein will mean the recombinant form). Inthe binding tests, both SpA and mod-SpA were biotinylated according tomeans known in the art, for use in an enzyme linked immunoassay (i.e.,EUSA) and flow cytometry.

As shown in Table I, within this limited panel of monoclonal IgM, themod-SpA (without Fc binding activity) was bound by 15/16 V_(H) 3, butnone of the 7 V_(H) 1, 7 V_(H) 4, 1 V_(H) 5 and 1 V_(H) 6 proteinstested. Among monoclonal IgG, mod-SpA bound only 2/6 V_(H) 3 (GER andSFL), but none of the 4 V_(H) 1, 1 V_(H) 2, and 1 V_(H) 4 proteinstested. Therefore, these data demonstrate that using Ig examples of theV_(H) families, V_(H) 1, V_(H) 2, V_(H) 4, V_(H) 5 and V_(H) 6 proteins,did not express SpA binding capacity, because SpA binding was limited tocertain Fab that contain V_(H) 3 H chains (X², P<0.001). Moreover, SpAbinding was present in Ig from at least four V_(L) families, includingboth kappa and lambda L chains and SpA binding activity was notrestricted to antibodies with a single conventional antigen bindingactivity. Also, H chains denatured and reduced in Western immunoblotanalysis cannot bind SpA.

This data supports the impression that the V_(H) binding site for SpA isa conformational component, which requires L chain for expression eventhough L chains may not be directly involved in SpA binding. Inaddition, although this panel contained antibodies with a variety ofconventional antigen binding actities, both autoreactive (RF, anti-DNA,anti-Sm) and anti-bacterial (anti-Haemophilus and anti-pneumococcal)antibodies, SpA binding activity was not restricted to antibodies with asingle conventional antigen binding activity. Further, it should benoted that binding to the "alternative binding site" of SpA may be lesscommon in the products of more mature B cell populations as a muchlarger proportion of the V_(H) 3 IgM (17/19) than V_(H) 3 IgM (2/7)bound the modified SpA (X², P<0.005). A partial compilation of this datais shown in Table I in the left hand columns.

                                      TABLE 1    __________________________________________________________________________    Structural and functional properties of monoclonal lg proteins.    V.sub.H 1             V.sub.H 2                     V.sub.H 3 V.sub.H 4.sup.+                                       V.sub.H 5                                                V.sub.H 6    SPA    V.sub.L                SPA                   V.sub.L                          SPA                             V.sub.L                                  SPA                                     V.sub.L                                           SPA                                              V.sub.L                                                  SPA                                                     V.sub.L    __________________________________________________________________________    LgM    And -  κ3  Buc  +  κ1                               Cor                                  -  κ3                                       A224*                                           -  λ                                                L16*                                                   -  λ    Bor -  κ3  Cha  +  κ1                               Far                                  -  λ    Gil -  κ4  Chr  +  κ1                               Gre                                  -  κ3    Jan -  κ3  Dau* +  κ3                               Les                                  -  κ3    Kas -  κ3  Erik +  κ3                               Odo*                                  -  κ3    Pal -  κ3  Glo  +  κ3                               Ore                                  -  κ3    Sic -  κ3  Hea  +  κ3                               Ple                                  -  κ3                     Kim 4.6*                          +  κ3                     Lay  +  κ1                     Pom  +  κ3                     Riv  +  κ3                     Sim  +  κ4                     18/2*                          +  κ1                     4B4* +  κ4                     591* +  λ                     loh  -  κ4    pG    Fine*        -  κ2             Cess*                -  λ                     Ger* +  NT                               Cla*                                  -  κ1    Heb*        -  λ  SFL* +  κ3    Hou*        -  κ1  Cal* -  λ    Long*        -  λ  Do*  -  κ1                     Fnz* -  λ                     Mag* -  NT    __________________________________________________________________________

Regarding the site on SpA which is responsible for V_(H) 3 binding, thedata set forth in Example I below demonstrate that the "D" domain of SpAcontains an Ig Fab binding domain. This domain may itself be utilized asthe sAg for use in the vaccination methods of the invention.

To explain, it is known that SpA has 5 independent, homologous domainswhich can bind Ig (commonly referred to as domains A, B, D, E and X).However, it has not been known which of these regions, if any, possessFab (as opposed to Fc) binding activity. The work described in Example Idemonstrated for the first time that domain D not only will bind Ig Fab,but will also preferentially bind Fab of V_(H) 3 restricted Ig. As aresult, SpA and this domain of SpA can be used as a carrier or adjuvantin an immunization protocol to specially enhance the production of V_(H)3 restricted antibodies by a patient.

iii. Correlation of V_(H) region sequence with SpA binding activity.

All available V_(H) sequence data from the above-described panel werecompiled to determine whether Ig that bind mod-SpA have conservedstructural motifs (Table II, right hand column). Sequence analysisconfirmed that the V_(H) regions of Ig that bind SpA share considerablehomology in the framework (FW) regions. However, the composition of thehypervariable regions vary greatly, because these H chains express V_(H)regions that represent rearrangements of at least five different V_(H) 3germline genes.

Immunization with a conventional epitope often induces antibody cloneswith the same (or very similar) V genes within a family, which oftenshare conserved sequences in the hypervariable loops of V_(H) and/orV_(L) regions that may be intimately responsible for antigen binding. Incontrast, a V_(H) specific superantigen would be predicted to interactwith diverse members within a V_(H) family, which do not necessarilyshare sequences within the hypervariable loops. Therefore, conservationof V_(H) FW residues in Ig with SpA binding likely reflect the B cellsuperantigen functional capacities, because the interaction is outsideof the conventional antigen binding pocket.

In Table II.A the amino acid sequences of H chains from Ig reactive withmod-SpA are displayed. The amino acid sequences of H chains forevaluation of binding to mod-SpA by immunoassay are displayed (all areV_(H) 3 region derived). The single letter code for amino acid residuesis used, and # represents a pyrrolidonecarboxylic acid residue, X isundetermined, and * is a space. The IgM proteins 4B4, 18/2, KIM4.6 andL16 directly derive from the germline configuration of V_(H) genesegments, and these genes are listed under GL V_(H). SB5/D6 and ED8.4are anti-hameophilus influemze PS cell lines. The IgM proteins (LAY,POM, RIV, BOR, KAS and SIE, and IgG protein SFL, are circulatingmonoclonal antibodies from patients with lymphoproliferative syndromes.Huabl4-3 and Huab 2-3 are antipneumococcal capsular polysaccharide type3 cell lines. The conventional antigen binding activity of V_(H) 3antibodies is listed under Ag⁺. Relative reactivity with modified-SpA isdepicted as OD₄₀₅ 0.100-0.400+0.401-0.800++, 0.801-1.200+++. NTindicated that binding for a sequence was not tested.

    TABLE 2A      - Human V.sub.H Regions and SpA Reactivity.sup.1      ##STR1##      10/2V.sub.x                                                             S    A      26DNAIgN++EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMS**WVRQAPGKGLEWV      ##STR2##      KD8.4HIbPS1gM+++-----------------------------------**--L-P-------------**     ---R------------      ---E-------V------V----------GYG************KDVW      Nusb14-3PS31gM+++----V-------------------------T-R-R**----------V---R-NS*    *     -S--D-----E----      -----A--------------------FDS*********************-----------      SFLRFIgG++----V------------------------TKYX--**-------------AFIYR**--INR     ------M----      -----X----XXX----------L---A-DAG-KVERSV      KIN.sub.4.G1-9111DNAIgM++Q---V-----V----R----------------G-H**----------     ---AVI-Y**--SHK-----------      ---------------------------A-VRKY---S-***-YYYYGMDV-----T-----      RIVRFIgN++++----V-----V----S---------------F--H**-------------AVM-Y**--DN    K     --V--------      -----------------------L---A-LSTAAS-FTFDTYGMD-**-----T-------      LAYRFIgM+++A-----------------------------AS---**-------------AWKYE**N-NDK    H     -----N----      ---ND------------Q---S-I---ARDAGP-VSPTF********FAM-----------      PONRFIgN++++-------------------------------S---**-------------AWKYE**N-ND    K     -----N----      ---ND-------L----Q-----L---ARDAGP-VSPTF********FAHY----------      Nusb2-3PS3IgM++++----V-------------R-----------AS-VH**WV---S-------GR-RSK    A     -A--A-------      ----D----A-------KT---------GHPLTTVTTP*******----------------      SB5/D6HIhPS1gA1++----V-------K-----------------NAH-N**----P--------GR-KTK    T     --T-D--AP------      ---ND------------KT---------TGGGV************G      4D49-1SmIgN+----V-------K-----------------NAW--**-------------GR-KSKTD--T     -D--AP------      ----D------------KT---------TDSLPPHRV*************-----------      ##STR3##      -----------------------L---ASSGAGNGLPSLDY*********-----------      SpA3-02SpAT++++-K----------------------------H---**--------------D---**-     ---------------      -----------------------L---ANSGAGWGLPSLDY*********-----------      SpA3-37SpAT++++-K----------------------------H---**-------R------D---**-     ---------------      -----------------------L---ANSGAGWGLPSLDY*********L----------      SpA3-13SpAT++++-K----------------------------H---**-------R------D---**-     ---------------      -----------------------L---ANSGAGWGLPSLDY*********-----------      SpA3-15SpAT++++-K----------------------------H---**-------R------D---**-     ---------------      -----------------------L---ANSGAGWGLPSLDY*********-----------      SpA3-16SpAT++++-K----------------------------H---**-------R------D---**-     ---------------      -----------------------L---ANSGAGWGLPSLDY*********-----------      SpA3-18SpAT++++KL-K--------------------------R---**-------R------D---**-     ---------------      -----------------------L---ANSGAGWGLPSLDY*********-----------      SpA3-39SpAT++++KL-K--------------------------R---**-------R------D---**-     ---------------      -----------------------L---ANSGAGWGLPSLDY*********-----------      SpA3-33SpAT++++KL-K----------VP-----K----P--N-G--**--------------S---**-     -D-------------      -----A--S------------------ARDAWDAFDI*************-----M-----      SpA2-07SpAT++++VKL-EQ------------------------H---**-------R------D---**-     ---------------      -----------------------L---ANSGAGWGLPSLDY*********-----------      SpA1-30SpAT++++VKL-EQ------------------------H---**-------R------D---**-     ---------------      -----------------------L---ANSGAGWGLPSLDY*********-----------      ##STR4##      V---D-QS-V----S---T--------GTRDY***********      CA4/PRP-13 HIbPSIgG2-----V-------T----------------TFNTY-HN--------------S     --R**-SDYI--P--------      ----TP--PV----T---D--------A-      ##STR5##      -TT-E-TS-A-MEVS---S----L---AREGRRHAINP*******--Y-----------      KASV.sub.H                                                              P    *      1RFIgM-#---VQ--AEVKK--S-VKVT-K---G------I-**-------Q----MGG-I-QAN--QRFQ-     -V-      -TA-E-T--A-MELR---SD---M---A-EGYGD--RP*******--F-----------      SIEV.sub.H                                                              K    *      1RFIgM-#---VQ--AEVKK--S-V-VT-KT--Q---G-TFS**-------R-----GSPA-V-      V-LKP-F-QA-MELVN-FN--G-----AREWKGQVNVNP******--Y----V------      CRSSV.sub.H                                                             *    *      2?IgM-Q-N-R---PA--KATHT-T-T-TF--LSVNTRG--VS-I--P---A---LAR-D--GT-LET-L-      --K-T---QVV-KVTNMDPA---T---ARM--TMVRKVMITSNAF*-I-----M-----      LESV.sub.H                                                              *    *      4RFIgM-QVQ-QQW-A--LK-S-T-S-T--VI-GP--G-YN-**----P----P--IGE-N-R-T-NP-LTS     -V-      M-L-T---QFS-KLT-VT-A---V--LAR-PCZA-CTDDAPQA*YFQH----      A224V.sub.H                                                             L      5PolyIgM-----VQ--AEVKK--E--KI--KT--TS-TN-WIG**----N-------MG-YP**GDSG-R-S    P     -F--QV-      --A-K-IS-A---WS----S---M---AR      L16V.sub.H                                                              Y    Y      6PolyIgM-Q---QQ--P---K-SQT-S-T--I--DSV--NSAAHN-I--S-SR----LGH-KHYND--V--     -S-I-      -NF-T---QFS--LN-VTP----V---ARELGDAF***********-I-----M-----     .sup.1 The amino acid sequences of monoclonal Fab and antibodies are     displayed with their SpA reactivity. The single letter code for amino aci     residues is used, and # represents a pyrrolidonecarboxylic acid residue,     is undetermined, * is a gap without a residue and - indicates the same     amino acid as in the top sequence is used.     .sup.2 If these VII regions are directly encoded by VII gene segments in     their germline configuration, these genes are indicated.     .sup.3 If the antibody was initially identified based on binding to a     conventional bacterial or self antigen, it is indicated. RF, rheumatoid     factor. DNA, antiDNA binding. Hib PS, antibody activity to the capsular     polysaccharide of HAemophilus influenzae type b. PS 3, antibody activity     to the capsular polysaccharide of S. pheumoniae type 3. 7, no known     binding activity. Poly, polyreactive.     .sup.4 The relative binding activity to SpA that has been modified to     remove Fe binding activity, but retains Fab binding activity is displayed     Relative reactivity with modifiedSPA by direct binding ELISA is depicted     under modSpA, as OD.sub.405 0.100-0.400 +, 0.401-0.800 ++, 0.801-1.200     +++, > 1.200 ++++. NT, not tested.     .sup.5 These are intact IgM and IgO antibodies with significant Mod SpA     binding activity. The IgM 4B4, 18/2, and KIM4.6 are from B cell lines tha     use germline configuration VH gene segments. The IgM proteins, LAY, POM,     RIV, BOR, KAS AND SIE, AND IgO, SFL, are circulating monoclonal antibodie     from patients lymphoproliferative syndromes.     .sup.6 These monoclonal Fab were isolated from a combinatorial library     based on SpA binding capacity.     .sup.7 These are monoclonal VH3 antibodies that are devoid of SpA binding     activity.     .sup.8 These are nonVH3 monoclonal antibodies that are devoid of SpA     binding activity.

Table II.B depicts the DNA sequences of V_(H) regions of SpA bindersfrom combinatorial libraries (discussed further under Step 2, below).The amino acid sequences of Table II.A as well as the nucleotidesequences of Table II.B are set forth in the Sequence Listing herein asSEQ. ID. NO: 1 through SEQ. ID. NO: 48.

    TABLE 2B      - DNA sequences of V.sub.H regions of SpA binders from combinatorial     libraries.sup.1      ##STR6##      ##STR7##                                                                              S     pA3-08....................................................................    .      ........................................                                                                              S     pA3-13....................................................................    .      ........................................                                                                              S     pA3-15....................................................................    .      ........................................                                                                              S     pA3-16....................................................................    .      ........................................                                                                              S     pA3-18GTGAAACTG..CGAG.....................................................    .      ........................................                                                                              S     pA3-18GTGAAACTG..CGAG.....................................................    .      ........................................                                                                              S     pA3-37....................................................................    .      ........................................                                                                              S     pA3-33GTGAAACTG..CGAG..............C................T.C..................A    .      .C....C..T.A.T...G......................                                                                              S     pA2-08...........................ACC...........G..........................    .      .............T..........................                                                                              S     pA1-30GTGAAACTG..CGAGC..............................T.C..................A    .      .C....C..T.A.T...G......................                                                                              S     pA3-29.............................C......................................    .      ......C..T.ATT..........................                                                                              S     pA1-14.............................C..A..T................................    .      ......C..T..TT.CTGG...CA............A...                                                                              0     -19.............................C.........................................    .      ...........C.TT........A................                                                                              V     H26CG.....C.G...T................C........................................    .      .............T..........................      ##STR8##      ##STR9##      SpA3-08..................................................................    .      ........................................      SpA3-13.......G..........................................................    .      ........................................      SpA3-15.......C..........................................................    .      ........................................      SpA3-16.......C..........................................................    .      ........................................      SpA3-18.......C..........................................................    .      ........................................      SpA3-39.......C..........................................................    .      ........................................      SpA3-37.......C..........................................................    .      ........................................                                                                              S     pA3-33...........G...............AG........CA.......A...T.....C..C..C.....    .      .....................CG........T.A......      SpA2-08...........C................C........GT.................C..C......    .      A....................CG.................      SpA1-30.......G..........................................................    .      ........................................      SpA3-29...........G................G.................A..C......C..C......    .      ....G..................T................      SpA3-14................T..........CG.....ACA.TGA...GA....A...AG...C..G...    .      A....................CG..........C......      0-19.....C..................G...T.G.AT.GTATGA...AAC..CT.AG..C............    .      A...........T........C...G.......C......      VR26C...........G................C........GT.................C..C.G......    .      ....................T...................      ##STR10##      TTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCC      SpA3-08...................................................TCCAGCGGCGCGGGA    T      ......................................      SpA3-13...................................................TCCAACGGCGCGGGA    T      ......................................      SpA3-15...................................................TCCAACGGCGCGGGA    T      ......................................      SpA3-16...................................................TCCAACGGCGCGGGA    T      ......................................      SpA3-18...................................................TCCAACGGCGCGGGA    T      ......................................      SpA3-39...................................................TCCAACGGCGCGGGA    T      ......................................      SpA3-37...................................................TCCAACGGCGCGGGA    T      ......................................                                                                              S     pA3-33C..................................TC.G............AGAGATGCATGGGATGC    A      .......A..C.......................      SpA2-08C....GC.......T...........G........TG.G...........AAGACCCGAGTTAGGA    G      TAACCCCCTCTG.G.................................      SpA1-30................................................... TCCAACGGCGCGGG    A      ......................................      SpA1-29C.........................G........TG.G..........GTAGAGGATACAGCTAC    C      ..A..G...AC.............      SpA1-14C.....C.......T...........G........TG.G...........AAGAGCCGAATTCCGA    C      CTAACCCCCCTCC...G.................................      0-19C.....................T...G........TG.C...........AAAGGATACCAGTTG      T......................................      VII26CC.........................G.........G..............AAA     .sup.1 These DNA sequences of VII regions were obtained from monocional     Fab with SpA binding activity. The germline gene VII26c is included for

To further refine a model of a V_(H) FW defined SpA binding site, SpAbinding activity may be correlated with single V_(H) region mutations.In particular, FW sequences may be correlated sequences. Therefore, FW1and/or FW3 components can be critical for binding of conventionalantigens, and conserved FW motifs have also been shown to be criticalfor binding of a T cell superantigen. Significantly, amongst members ofa V_(H) family, the sequences in FW1 and FW3 have limited variabilityand the conservation of residues in FW3 within members of a family hasbeen proposed to provide initial family associated constraints foraffinity for specific epitopes.

On the H chain, the FW1 and FW3 regions represent a large surface thatis highly solvent exposed, and which is not directly in contact with theL chain. Taken together, these data suggest that it is highly possiblethat FW residues form the contact region for the alternative bindingsite of SpA, but that constant-domain regions may still affect theinteraction. For these reasons, mutagenesis experiments can beinformative in the determination of whether FW3 (with or without FW1)directly contributes to the contact site of a B cell superantigenbearing in mind that amino acid changes in non-contact residues cancause remote conformational changes can affect binding affinity.

Step 2. Assessment of V region diversity on binding activity using abacterial expression system.

i. Construction of a combinatorial library.

Optionally, the binding site on Ig for the candidate antigen can belocalized and characterized. Useful to this end is the pCOMB3 phagemidvector constructed at Scripps Clinic, La Jolla, Calif. (laboratory ofDrs. Dennis Burton and Richard Lemer). As depicted in FIG. 1, pCOMB3will efficiently clone and express, separately or in combination, humanIgH and L chains.

The vector is designed to create a fusion protein between the V_(H)fragments and gill (the minor coat protein of the M13 phage), which canbe arranged on the surface of M13 phage that contain the encoding Vgenes (disclosed in Barbas, et al. (1991) Methods Enzymol. Z:119-124,which is incorporated herein). The gene encoding the M13 portion of thefusion protein can also be excised with SpeI and NheI and, afterre-ligation, the transfected bacteria secrete Fd fragments V_(H) -CH1and V_(L) -CL1).

The pCOMB3 vector (and a related pCOMB8 vector) are not at this timebeing made available on an unrestricted basis. However, a comparablevector in a modified lambda gt11 phage is available commercially fromStratacyte of La Jolla, Calif. Other comparable vectors for the surfacedisplay of antibody Fab on a filamentous phage that grows in an E.Colihost are known may be available from William Huse, IXSYS, Inc. (SanDiego, Calif.), Cary Queen, Palo Alto, Calif. (described in Chang, etal. (1991) J. Immunol. 10:3610-3614), and at the laboratory of GregWinter, Cambridge University, England.

In FIG. 1, the pCOMB3 vector is shown in surface display and solubleFab-producing forms. The XhoI and SpeI sites are provided for cloningPCR-amplified H chain Fd sequences. The SacI and XbaI sites are providedfor cloning PCR amplified antibody L chains. The PelB leader sequencesaid transcription and processing in the E.Coli host, with reading framesmaintained. The parent sequence of the phagemid vector, pBluescript,which includes CoIE1 and F1 origins and a β-lactamase genes, haspreviously been reported. Digestion of pCOMB3 encoding a selected Fabwith SpeI and NheI allows for the removal of the gill fragment, a M13protein. Since SpeI and NheI produce compatible sticky ends, thedigested vector may be re-ligated, yielding a phagemid that producessoluble Fab.

FIG. 2 illustrates the composition of the pCOMB3 vector and the proposedpathway for Fab assembly and incorporation into the phage coat.Expression of Fd/cpIII (H chain and M13 protein fusion product) and Lchain is controlled by Iac promoter/operator sequences. The chains aredirected to the periplasmic space by pelB signal sequences which aresubsequently cleaved. The H chain is anchored to the membrane by thegIII fusion, whereas the L chain is secreted into the piroplasm. The twochains are then assembled on the bacterial membrane. The Fab molecule isthen incorporated into the M13 phage coat during exusion of the phagethrough the membrane via the cpiII segment (taken from the Barbas, etal. publication cited supra).

ii. Application to SpA.

As used with SpA, the vector was used to create a polyclonalcombinatorial expression library from the peripheral cells of a healthyvolunteer. Combinatorial libraries with gamma H chains were chosen forthe determination of the basis of SpA binding for the followingreasons: 1) This vector creates Fab without Fc regions so the screeningmethods are simplified; 2) with multiple V region primers and provenmethods huge libraries (>10⁷ H chains and >10⁷ L chains) can be createdto facilitate a highly rigorous examination of the effect of structuraldiversity on binding; 3) the vector facilitates the efficient creationof hybrid molecules with different H-L chain pairing, so the effects onbinding can be determined; 4) the use of a gamma library allows theexamination the effects of somatic replacement mutations on binding; and5) these libraries offer an optimal source of cloned V genes for sitespecific mutagenesis experiments.

Antibody clones within the library that bind to SpA were selected andamplified using PCR according to means well known in the art. Thisprocess is the experimental equivalent of in vivo clonal competition forantigen, due to selection for the clones with the greatest bindingaffinity. Beginning from a library in which 0/30 random clones boundSpA, after three rounds of selection were performed wherein 27/30 randomclones bound SpA strongly, as detected by incubation of bacterial liftswith ¹²⁵ I-SpA (see. FIG. 2), which detects only clones with highaffinity for SpA.

Specifically, after induction with IPTG, colonies were lifted ontonitrocellulose filters, and positive clones were identified by using ¹²⁵I-SpA (0.1 mc/ml; ICN Radiochemicals, Irvine, Calif.), usingautoradiography. Positive colonies appear dark on FIG. 3. Randomlyselected colonies from the original library are depicted at the upperleft of the Figure. Colonies from the first round of panning are shownupper right Phage input was 2×10¹¹ cfu, and 9.5×10⁵ cfu were elutedafter the first panning (% yield=4.75×10⁻⁴). Colonies from the secondround of panning are depicted at bottom right Phage input was 2×10¹¹cfu, with 5.3×10⁶ cfu eluted after the second panning (%yield=2.65×10⁻³). Colonies from the third round of panning are depictedat bottom left. Phage input was 2×10¹¹ cfu, and after the third round ofselection 9.5×10⁵ cfu were eluted (% yield=4.75×10⁻³).

It should be noted that the phagemid clones express only abbreviatedgamma chains, which is devoid of the gamma second and third domains, sothey do not express the Fc region identified by non-modified SpA. Afterprolonged exposure, in the original library none of the 30 dones bound¹²⁵ I-SpA, while after one round of selection 1 weak binder of 30 cloneswas detected. After the second round of selection 7/30 clones haddetectable SpA binding, and after the third round 27/30 clones bound SpAstrongly.

Referring to Table III, it is shown that the composition of theoriginal, unselected combinatorial library was compared to the librariesafter three successive steps of selection with SpA and expansion. From17 to 24 clones were randomly selected from each of the libraries afterthe gIII gene was excised. H chains and κ and λ L chain expression wasdetected by conventional sandwich ELISA. In addition, replicate slotblots with 200 ng of purified plasmid were probed with biotinylatedoligonuceoties and a nonisotopic detection system (Tropix, Bedford,Mass.). For these studies control H chain genes of known sequence 7E(V_(H) 1-gamma), 30P1 (V_(H) 4-mu)! were used. To detect γ chainexpression, the antisense sequence, GTCCTTGACCAGGCAGCCCAGGG, designatedgamma-CH1, from the first domain was used. Detection of V_(H) familiesused sense oigonucleoaides from the third framework regions; of V_(H) 1,5'TACATGGAGCTGAGCAG(CG)CTGAGAT 3', designated V_(H) 1-FW3, of V_(H)3,5'TATCT(TG)CAA(GA)TGA(AG)CAG(TC)CTGA(ACG)A 3', designated V_(H) 3-FW3,of V_(H) 4, 5'TCCCTGAAGCTGAGCTCTGTGACCG 3', designated V_(H) 4-FW3.Clones designated ?V_(H) were identified by the gamma probe, but by noneof the three V_(H) family probes.

                  TABLE 3    ______________________________________           Original library                    SpA       SpA      SpA           (unselected)                    1         2        3           (N = 24) (N = 20)  (N = 20) (N = 13)    ______________________________________    H-L pair 15/24      11/20     14/20  13/17             (63%)      (55%)     (70%)  (76%)    SpA Binders             4/24       5/20      14/20  13/17             (17%)      (25/%)    (70%)  (76%)    gamma    19         19        20     13    V.sub.H 1             5/19       1/19      0/20   0/13             (26%)      (5%)      (0%)   (0%)    V.sub.H 3             5/19       11/19     19/20  13/13             (26%)      (57%)     (95%)  (100%)    V.sub.H 4             5/19       6/19      1/20   0/13             (26%)      (32%)     (5%)   (0%)    ?V.sub.H 4/19       1/19      1/20   0/13             (21%)      (5%)      (0%)   (0%)    ______________________________________

These data highlight that not all of the clones in the original libraryexpressed both functional H and L chains, but this proportion increasedby the third round of SpA selection. Data from the EUSA method ofdetection was more sensitive, and detected more SpA binders than themembrane lift method (displayed in FIG. 2). Significantty, we found that17% of clones in the unselected library bound SpA, but with weakaffinity (as measured by apparent K_(D)). After one round of selectionthis increased to 25% SpA binders. After the second and third round ofselection this stablized at approximately 70%, and K_(D) measurementsdemonstrated that there was an increase in average affinity amongst SpAbinders with each round of selection. Of note, every SpA binderexpressed both H and L chains, and used V_(H) 3 encoded H chains,although both κ and λ L chains were used by different clones. This studyrigorously confirmed the findings from the survey of isolated monoclonalIg, which indicates that the "alternative binding site" of SpA is V_(H)3 restricted.

                  TABLE 4    ______________________________________    Apparent affinities of isolated lg and pCOME3 clones    from SpA3 library.              L CHAIN  I.sub.0.5 (10.sup.-3 M)                                 K.sub.D (10.sup.-3 M)*    ______________________________________    Purified lg    Poly lgM    poly       58.0      38.0    Poly lgG Fab                poly       13.0      16.0    RIV (Mono lgM-V.sub.H 3)                V.sub.χ III                           140.0     45.0    SpA3 Library    SpA3-18     λ   2.2       5.5    SpA3-08     λ   4.4       12.0    SpA3-37     V.sub.χ III                           3.2       9.2    SpA3-22     χ      8.7       6.6    SpA3-40     λ   11.0      18.0    SpA3-02     V.sub.χ III                           13.0      28.0    SpA3-39     λ   12.0      26.0    SpA3-33     V.sub.χ III                           22.0      46.0    SpA3-13     V.sub.χ III                           81.0      29.0    SpA3-16     V.sub.χ III                           24.0      32.0    SpA2 Library    SpA2-13     λ   45.0      76.0    SpA2-09     χ      6.7       3.7    SPA2-14     χ      9.8       23.0    SpA2-05     χ      7.0       12.0    SpA2-18     χ      4.8       24.0    SpA2-01     χ      20.0      16.0    SpA2-07     χ      120.0     505.0    SpA2-06     χ      11.0      14.0    SpA2-17     χ      16.0      20.0    SpA2-19     χ      1.3       6.7    SpA2-02     χ      ND        48.0    SpA2-04     χ      12.0      8.7    SpA1 Library    SpA1-14     χ      17.0      11.0    SpA1-19     χ      0.5       5.1    SpA1-29     λ   19.0      49.0    SpA1-30     λ   50.0      61.0    ______________________________________

An inhibition ELISA was used to compare the apparent affnities of clonesfrom the SpA3 library, with a monocional IgM V_(H) 3 protein and apolyclonal IgM and IgG from adults; apparent affinity (K_(D)) wasdetermined per the method disclosed in Friguet, et al. (1985) J.Immunol. Methods 77:305-319. These data (shown in Table IV) illustratethat the clones from the SpA3 library all have high affinity for theselecting antigen, SpA. Sequence comparison indicated that 5 of the6V_(H) sequences are likely clonaily derived, while the clone SpA3-33represents an independent rearrangement, and likely derives from adifferent germline V_(H) 3 gene (deduced amino acid sequences areincluded in Table II.A).

They are closest to the V_(H) 26c germline gene with less than or equalto 94% homology at a DNA level, which likely suggests that the truegermline sources of these genes have not yet been reported. Data fromTable III suggest that after three rounds of selection only clones withthe strongest SpA binding affinity are represented, which explains why5/6 V_(H) clones are nearly identical. These clones use both κ and λ Lchains. The binding affinity of these H chains for SpA when they useidentical L chains may also be measured, so the effect of each aminoacid substitution on SpA binding can be determined.

An additional source of non-diversified V gene segments may be developedfrom amplification and cloning of a genomic DNA library and >100 clonesV_(H) 3 genes from the same individual as the source of the previouslydescribed, peripheral lymphocyte library. To verify the integrity ofthis library, the sequences of four random clones are determined (here,W4, W8, W 11, and W12 included in Table II.A) representing new V_(H) 3germline genes. These V gene segments will have CDR3 and constantregions granted by overlap extension PCR, prior to insertion andexpression in pCOMB3, for testing of SpA, or other candidate antigenbinding.

The pCOMB3 vector was also used to clone the V genes expressed in ahuman B cell hybridoma line, designated CA4 (obtained from Dr. AlexLucas, Oakland Children's Hospital). When these genes were cloned intothe pCOMB3 vector, the resultant construct, termed PRP-12, was shown tohave the same binding specificity for the capsular PS of Haemophilusinfluenzae, type b (Hib) as the parent cell line (V_(H) sequenceincluded in Table II). By inhibition ELISA according to means well knownin the art, the parent IgG cell line, CA4, has an apparent K_(D) for HibPS of 2.4 X1⁻⁹ M, while PRP-12, the pCOMB3 clone that expresses these Vgenes, is 2.5×10⁻⁸ M. The lower affinity of PRP-12 for ligand, comparedto the parent CA4 line, may be partly due to receptor valency. The Vgenes from this cell line were selected for further study for severalreasons i) CA4 has binding activity for a conventional antigen, ii) thisantibody uses a V_(H) 3 H chain, iii) the parent cell line produced anantibody that is a V_(H) 3 antibody of known sequence without SpAbinding activity, and the bacterially expressed antibody also sharesthis property (as described above) and, iv) iii) the V_(H) regioncontains a few highly unusual amino acid residue substitutions in theframework regions, which have not been previously described. Thereforethe PRP-12/CA4 clone can be used to determine the effect of single pointmutations on both a conventional and a superantigen binding capacity.

Step 3. Confirmation of the sAg activity of a candidate sAg ininteractions with whole Ig bearing cells.

To confirm the sAg activity of a candidate antigen in interactions withIg bearing whole cells, binding percentages can be determined using flowcytometric analysis. This method is described below in an example usinglymphoid cells having IgM, IgG and/or IgD which are known to use asignificant proportion of the V_(H) 3 heavy chains (demonstrated inexamples above to be the region which most prevalently binds the SpAsAg). it should be noted that this approach, and the resulting data, isalso useful in determining whether particular sAg's can be used aslymphocyte phenotypic markers. Further, once the normal range of bindingfor a particular sAg is determined, the methods described below can beused to determine if variations from that binding pattern have occurredin a given B cell population.

For example, using the method below it has been determined that 14-50%of B lymphocytes in a healthy human individual will bind to the V^(H) 3restricted site on SpA. ft is known that a B cell malignancy (such as alymphoma or leukemia) may be present if a large or dominant monoclonal Vcell population is detected in the blood or in tissue samples taken froma patient. Therefore, in the presence of relevant diagnostic signs knownto those skilled in the clinical arts, very low (e.g., 0-5% ) or veryhigh (e.g., 80% or more) Fab-mediated binding of SpA by B lymphocytesfrom a human patient could indicate the onset or existence of amalignancy or the outgrowth of abnormal oligoclonal B cell populationsin a patient who is suspected of having or being susceptible todeveloping a B cell malignancy.

As a representative example, the binding specificities of SpA, mod-SpAand Protein G (obtained commercially from Pharmacia LKB Biotechnology,Piscataway, N.J.) were compared. To perform the study, mod-SpA andProtein G were bictinylated according to means well known in the art andincubated with a suspension of human (mononuclear) tonsillar cells(MOAB's). Binding of mod-SpA and Protein G thereto was detected by flowcytometry; it will be appreciated that other means for detecting bindingby a sAg or candidate sAg other than flow cytometry could be employed.It will also be appreciated by those skilled in the art that lymphocytesfrom peripheral blood and other lymphoid tissues, as well as lymphocytesfrom various species of interest could also be used in this analysis;for reference in this regard, analysis was also performed usingumbilical cord blood (which is enriched in V_(H) 3 B cells compared toadults).

As shown in FIG. 4, the results of this analysis for Protein G are shownin the left column, for SpA in the middle column, and for mod-SpA in theright column.

The percentages of cells reacting with bacterial antigen only (upperleft quadrants), Moab only (lower right quadrants) or both bacterialantigen and Moab (upper right quadrants) are presented. The top rowdisplays dual staining with a pan-T cell market (anti-CD3). None of thebacterial antigens bound to the CD3⁺ T cells. Instead, as shown in thesecond row, reactivity was restricted to the CD20⁺ B cell population.Subsets of IgM⁺ cells (third row), IgG⁺ cells (fourth row) and bothkappa⁺ and lambda⁺ cells (fifth and sixth rows) were also observed toreact with all three bacterial antigens. The quadrants were set usingcells reacted with the isotope controls and the streptavidin-PE reagent.Non-specific binding was less than 1% (data not shown).

On a percentage basis, the values for binding with cells from either theCD20 B cell population or which bear IgM, IgD, IgG, kappa or lambdasurface Ig are shown in Table V. These data were derived from dualstaining studies to determine the proportion of CD20 staining cellswhich also stain a biotinylated bacterial antigen.

                                      TABLE 5    __________________________________________________________________________           Cord               Cells Tonsillar                          Cells Protein G                                      SpA    Inhibitor           Binding               % Inhibition                     Binding                          % Inhibition                                % Inhibition                                      % Inhibition    __________________________________________________________________________    PBS    31        15    Human IgG           23  26%   9    40%   73%   64%    Fab (polyclonal    Human IgM           5   84%   4    73%   0%    71%    (Polyclonal)    Rabbit IgG           25  19%   15    0%   53%   24%    (polyclonal)    SpA3-08           7   77%   4    73%   0%     0%    (monoclonal)    PRP-12 26  16%   0     0%    0%    0%    (monoclonal)    __________________________________________________________________________    Tonsillar Cells Bearing:                   Protein G Binding                           SpA Binding    __________________________________________________________________________    CD20            9%     29%    IgM            24%     61%    IgG            24%     60%    K              25%     55%    λ       33%     59%    __________________________________________________________________________

                  TABLE 6    ______________________________________    V.sub.H Subset Expression after in vitro stimulatuion                     SRA     EBV    ______________________________________    % B6-IgM           30.0 +/- 8.4                                 10.6 +/- 4.0    % G6-IgM            1.4 +/- 0.6                                  5.5 +/- 2.1    Ratio (%B 6-1 gM/% G6-1 gM)                       21.4      1.9    ______________________________________

With respect to the MOAB's, these studies demonstrate that large subsetsof both IgM and IgG bearing cells bind modified SpA (which is without Fcbinding activity), likely due to the prevalence of V_(H) 3 H chains onthese lymphoid cells. SpA was bound by the largest proportion of Bcells, with a lesser proportion of cells binding Protein G. Mod-SpA wasbound by the smallest number of B cells. These differences in bindingare consistent with the prediction that Protein G is only bound by cellswith sIgG constant regions.

In contrast, SpA is bound to cells with the constant region of sIgGand/or certain V_(H) 3 determinants, while mod-SpA is bound only tocells with certain V_(H) 3 determinants. This model would, therefore,explain why SpA binds to a set of B cells that is roughly equal to thesum of the proportion of cells that bind to Protein G and mod-SpAseparately.

In this particular study, triple staining studies were performedaccording to means well known in the art to determine the basis forbinding of Protein G by 24% of IgM bearing tonsillar cells, becauseProtein G was believed to only bind to IgG. These studies demonstratedthat all IgM bearing cells from tonsil identified by Protein G alsocoexpress IgG. Significantly, mod-SpA identified 33% of IgM cells, butonly 20% of IgG bearing cells, which is in agreement with our bindingdata from isolated monoclonal IgG proteins. This observation confirmsdata from the evaluation of monoclonal Ig, by demonstration that alarger proportion of cells with sIgM than sIgG bind the alternativebinding site of SpA. Therefore, not only are the structural correlatesof binding of Fab to SpA unconventional, but the preferentialassociation of SpA binding in IgM is also in conflict with the acceptedparadigm of clonal maturation and expansion with isotope switch inresponse to a conventional antigen because after immunization with aconventional antigen, antibodies for the antigen are generally morecommon in the IgG pool than the IgM pool. In contrast, because withoutspecific immunization, SpA binds to more IgM than IgG, it is anunconventional antigen.

To document the Fab and V_(H) dependence of B cell staining with thebacterial antigens, the biotinylated antigens may be preincubated withdifferent Ig preps prior to cell staining. The data for protein G andMod-SpA binding to MOAB's are shown in Table V. These data illustratethat Protein G, which binds only to F_(c)γ, is inhibited by IgG but notpolyclonal IgM. Binding of mod-SpA by tonsillar B cells is betterinhibited by equal amounts (100 mcg/ml) of polyclonal IgM thanpolyclonal IgG, which is consistent with the impression that the averageaffinity of SpA binding in IgM is greater than IgG. Significantly,mod-SpA binding is not inhibited by preincubation with a monoclonalV_(H) 1 IgG which is consistent with the notion that mod-SpA binding isnot mediated via IgG constant regions, and that binding is mediated onlyvia certain Fab that do not include the V_(H) 1 family. In contrast, anFab product of the PRP-12 clone, which expresses the CA4 anti-Hib PSantibody, did not inhibit. The demonstration that a monoclonal Fab thatuses the appropriate V_(H) 3 H chain SpA3-08 can significantly inhibit Bcell binding, indicates that B cells bind to one or a very small numberof components on the alternative binding site of SpA. The sequenceencoding the SpA3-08 V_(H) region is included in Table II.B.

To that end, mod-SpA is preincubated with phosphate buffered saline(PBS) or with polyclonal human IgM or IgG, polyclonal rabbit IgG or theaffinity purified monoclonal Fab, SpA3-08 and PRP-12. in buffer for 1hour at 4° C. at a final concentration of 100 mcg/ml. Referring to TableVI, the proportion of the total B cells bound by mod-SpA is displayed,and the percent inhibition (compared to without inhibitor) is inparentheses. B cells were identified by the CD19 marker. In thesestudies, the biotinylated mod-SpA reagent was titrated to the end of theplateau of the binding curve for human B cells. Then prior to cellstaining, aliquots of mod-SpA were preincubated with either PBS(control), or potydonal human IgM or IgG, polyclonal rabbit IgG or theaffinity purified monoclonal Fab, SpA3-08 and PRP-12 The SpA3-08 wasisolated from a combinatorial library based on SpA binding (discussedabove under Step 2) and the PRP-12 expresses the genes encoding the CA4anti-Haemophilus influenzae type b antibody. The deduced V_(H) regionsof these antibodies are induded in Table II.A. The monoclonal V_(H) 3Fab, SpA3-08, inhibited >70% of binding to tonsillar and cord B cells,with greater inhibition of CD5-B cells than CD5+B cells. Only limitedinhibition occurred with the V_(H) 3 Fab, PRP-12.

Step 4. Confirmation of mitogenic activity of candidate sAg.

To confirm whether a candidate sAg can induce V_(H) restrictedlymphocyte mitogenesis, mononuclear cells from the species of interestare subjected to stimulation with the candidate and, for comparison,with a known mitogen (such as the Epstein-Barr virus).

Again, by example with respect to SpA, in vitro studies with umbilicalcord mononuclear cells are performed. This source was chosen because itcontains IgM bearing B cells and little or no IgG bearing B cells (sothe influence of native SpA's reaction with IgG Fc would be minimized).

Aliquots of purified B cells are stimulated in vitro either with anextract of Staphylococcus aureus, Cowans strain I (SAC), that containsSpA, or with the T cell independent B cell mitogen, attenuatedEpstein-Barr virus (EBV), B95-8, was used because the virus binds andstimulates B cells in proportion to their relative representation, andtherefore V gene usage in EBV stimulated cells has been shown to benon-biased. The Ig composition of these supernatants are evaluated forthe levels of two well characterized V_(H) associated idiotypes, G6 andB6, which identify subsets of V_(H) 1 and V_(H) 3 products,respectively.

Mononuclear umbilical cord supernatants are harvested after 8 days ofSpA/SAC stimulation and 28 days of EBV stimulation. The G6 and B6idiotypes are measured by immunoassay using standard curves with themonoclonal IgM, Sie and Glo, respectively (sources are described inSilverman, et al. (1988) J. Immunol Methods 82: 469-475, and Crowley etal. (1990) Mol. Immunol. 27:87-94, which references are incorporatedherein). All values are determined in mg/ml +/-SEM. Expression of theidiotypes on IgM after SAC/SpA and EBV stimulation are significantlydifferent (P<0.05).

The results (shown in Table VI) demonstrate that, compared to EBVsupernatant, SpA/SAC supernatant have a three fold enrichment in IgMthat bear a V_(H) 3 associated idiotype, and a four-fold depletion inIgM bearing a V_(H) 1 associated idiotype (κ² P<0.05). In contrast, Lchain isotope usage after SpA/SAC or EBV stimulation does not differsignificantly (data not shown). Therefore, the current data areconsistent with SpA binding studies, and this preliminary data indicatesthat at a protein level the "polyclonal B cell activator", SAC,stimulates B cell populations with preferential V_(H) 3 gene usage. Invitro activation with SpA appears to require co-stimulation from T cellsand/or cytokines, a mechanism analogous to the superantigen-propertiesof staphylococcal T cell.

Step 5. Determination of the earliest point in development in which Bcells bind Ig using SpA example!.

To best assess the possibility that SpA, or an equivalent superantigen,may be able to influence repertoire acquisition, the earliest stage of Bcell differentiation that certain B cell subsets may interact with theseagents, should be determined. Certain pre-B cells have both cytoplasmicH chains and low density surface expression of fully rearranged VDJ-muchain in association with a surrogate L chain. The functional capaity ofthis complex is unknown, but molecular modelling studies suggest thatthe pocket made up of folded hypervariable regions may be obscured inthese pre-B cell sig surrogates, while the region made up of the V_(H)FW1 and FW3 is surface exposed. While the V_(H) distribution of pre-Bcells from healthy individuals is unknown, at least 50% of therearrangements in fetal libraries from pre-B cells, and 40-60% humanpre-B cell leukemias use V_(H) 3 genes, therefore these genes arecommonly expressed in physiologic and pathologic examples of earlystages of B cell development. Moreover, these studies are important,because antigens with the ability to stimulate B cells at this earyproliferative phase would likely have much greater potential to skew theimmune response.

To determine whether SpA can interact with cells at this stage,microfluorimetric studies of pre-B cell lines are performed. Severalhuman pre-B cell lines are available from independent investigators forcorrelation of SpA binding and V_(H) expression (See Tsubata, et al.(1990) J. Exp. Med. 172:973-976; Rolink, et al (1991) Cell 66:1081-1094;and Nishimoto, et al., (1991) Proc. Natl. Acad. Science USA88:6284-6288). In addition, to isolate normal early lymphoid progenitorsfrom bone marrow, microfluorimetry can be used to sort CD34⁺ cells, or acommercially available anti-CD34 column (CellPro Inc., Bothell Wash.)can be used. In bone marrow, CD34 enriched populations have twosubpopulations-larger cells (i.e. increased SSC and FSC) that are CD10that represent myeloid cells, and smaller cells that are CD10+ thatrepresent lymphoid precursors. This latter populations of lymphoid cellscontain the majority of terminal deoxynucleotidyl transferase cells andinclude pre-B cells. And, although these cells represent less than 1% ofmarrow cells, adequate numbers of purified cells should be available totest the hypothesis that certain pre-B cells bind mod-SpA by a surrogateIg complex.

These samples are characterized for the expression of monocytic andmyeloid markers, and CD20 (a marker for more mature B cells). Then,binding of biotinylated SpA, modSpA and Prot G are assessed incomparison to CD19 and Sig markers.

To corroborate these studies, ¹²⁵ I-surface labelling of cells isperformed, followed by precipitation with superantigen, using publishedmethods (see, Nishimoto, supra). Briefly, cells are labelling and thenincubated with mod-SpA (or another microbial antigen), then washed andthe cells are lysed in the presence of digitonin and proteaseinhibitors. The precipitants are then sedimented and washed. Theprecipitated materials are run on reducing and non-reducing PAGE gels,to demonstrate the characteristic MW of the precipitating surrogate Igcomplex. The origin of the V_(H) region is also determined, byimmunoblotting of the precipitated complexes with the previouslydescribed anti-peptide antibodies to the mu constant region and Vfamilies. In addition, control precipitations may be performed usinganti-mu antibodies (as a positive control), and anti-kappa or anti-gammaantibodies (as negative controls).

Step 6. Cofactor requirements for in vitro stimulation with SpA.

To determine the cellular requirements for activaion by sAg (in thisexample, recombinant SpA), a series of in vitro stimulation studies willbe performed. In particular with respect to SpA, this is necessarybecause highly purified B cells are not stimulated by SAC, andsimilarly, B cells are not stimulated by recombinant SpA alone.Therefore, to determine the minimum requirements for cofactors toprovide the second signal during in vitro stimulation, activation withrecombinant SpA and control Ig binding proteins, in comparison with SACand other T cell independent B cell mitogens should be assessed.

To investigate the capacity for SpA and SAC to cause the selectiveproliferation of B cells, certain recently reported methods may beadapted to this end (see, Whisler, et al. (1991) Lymphokine CytokineRes. 10:1-6; and Boumpas, et al. (1990) 145:2701-2705). As described,mononuclear cells are first separated by a Ficoll gradient, then theyare first depleted of monocytes and NK cells by incubation using 5 MmL-leucine methyl ester HCL (Sigma) in serum free RPMI (tissue culturemedia). The cells are then washed and incubated twice with treated sheepred blood cells. To isolate B and T cell enriched fractions thenon-rosetting and resetting fractions are separated bydiatrizoate/Ficoll gradients, respectively. The sedimented rosetteforming ceiling from the first centrifugation are treated with isotonicNH₄ C1 to lyse the SRBC, and the cells are then analyzed with OKT3 (Tcell marker), CD19 (B cell marker) and CD16 (NK cells) to evaluatepurity. Residual contaminating cells can be removed by treatment withthe appropriate antibody and complement.

Proliferation studies are performed using formalinized SAC (Sigma) orStaphylococcal aureus Wood strain (that is devoid of SpA), the T cellindependent mitogens; EBV and phorbol esters (PMA), anti-IgM onsepharose beads, which will be compared with recombinant Ig bindingproteins, SpA (Calbiochem), mod-SpA or Protein G. Significantly, bythemselves, recombinant SpA and Protein G alone are devoid of mitogenicactivity, and earlier reports of mitogenic potential have subsequentlybeen ascribed to contaminants. While there is conflicting dataconcerning the mitogenic potential of soluble anti-human IgM, thereappears to be concordance that by proliferation criteria, as well as bybiochemical evidence of action (e.g. phosphorylation of tyrosinatedproteins and polymerization of cytoskeletal actin) that goat anti-humanIgM crosslinked to acrylamide beads is a more potent stimulant.

Even in the absence of class II molecules, T cell superantigens bound tobeads have been shown to be potent in vitro T cell stimulants,therefore, for a similar rationale, in vitro stimulation studies shouldbe performed to assess activity with the Ig binding proteins coupled tobeads. If mitogenic activity with a recombinant product is detectedwithout the addition of cytokines, control studies should be performedto evaluate for possible endotoxin contamination.

Cultures will be performed without the use of T cells, as studies withequivalent methods have demonstrated that recombinant IL-2 (10-20u/ml)(Cetus, Emeryville, Calif.) alone, or in combination with IL-4 (100u/ml)(Genzyme, Boston Mass.) can provide the "second signal" B cellproliferation. Available data suggest that proliferation does notrequire other cytokine factors, but the results of initial experimentswill determine the need for IL-1α, IL-6 and/or IL-7. Replicate culturescan be performed with 100 ul or 1 ml cultures of mononuclear cells at10⁶ cells/ml incubated with filtered supematants from attenuated, B95-8marmoset lymphoblastoid cells in 48 well polystyrene culture trays(Costar, Cambridge, Mass.) diluted 1:2 with fresh supplemented medium.All studies should be performed in replicates, with noninfected wells ascontrols.

Proliferation will be assayed using methods known to those skilled inthe art, by incubating the cells in medium supplemented with 1 uM ³H!-thymidine, with harvest onto glass fiber paper, and ³ H!-TdRincorporation determined by liquid scintillation spectroscopy. The timecourse of proliferation will be determined. Taken together, thesemethods will determine whether the stimulation of B cells by recombinantSpA has different requirements, or induces a different level ofactivation, than stimulation with EBV, PMA or by cross-linking ofantigen receptors by anti-Ig.

Step 7. Comparison of the repertoire induced by in vitro stimulationwith SpA to other polyconal activators.

Whether the subset of B cells that responds to in vitro stimulation witha sAg (here SpA) are restricted by their surface expression of V_(H)regions can also be evaluated, to determine if SpA causes a preferentialstimulation of V_(H) 3 B cells compared to unstimulated or mitogenstimulated controls. To determine whether various B cell mitogens inducesubsets of B cells with different V gene usage, methods to assess V geneexpression at both an RNA and protein level will be used. To assess thestimulation of different B cell subsets at an mRNA level, afterstimulation, 10⁷ cells are pooled, washed and sedimented, and the RNAextracted by the alkaline lysis-guanidium isothiocyanate method.Northern blots are performed using either labelled-FW oligonucleotidesor nick translated full length V_(H) family specific probes.Alternatively, the method of quantitative PR C described by Braun andcoworkers can be utilized.

Briefly, to determine the representation of V_(H) families, the RNA isused to make cDNA by using an oligo-dT primer and reverse transcriptase.V_(H) family specific FW1 primers and an anti-sense ^(J) H consensusprimer are utilized in application reactions, with quantitation by useof amplification with Taq polymerase and dNTP that include ³² P- α!-CTP.Samples are titrated so that the initial template represents >100 copiesof from each V_(H) family, with comparisons to cloned V gene standardsin plasmids. Equivalent volumes from amplifications using each of theV_(H) primers are then run on agarose gels, and stained with ethidiumbromide, then quantitated by counting in a scintillation counter. Asdescribed in the preliminary results sections, a large panel of clonedcDNA rearrangements for different V regions are available as positivecontrols for these studies.

For small scale purifications, the Ig from the supematants is purifiedby incubation with immunosorbent beads with anti-human Ig (Biorad,Richmond Calif.). In control studies, incubation of 10 ul of these cleanwashed beads with supernatant overnight at 4° C., binds greater than 15mg of Ig. Then, the beads are washed with 0.05% Tween-20/PBS, thenreducing buffer is added (2M and 8M urea), and the denatured Ig solutionis loaded onto SDS-polyacrylamide gels, for immunoblot analysis of Vregion usage in H and L chains. Therefore, by comparison of supernatantfrom cells stimulated with different mitogens, this approach shouldeffectively demonstrate the V region usage of responder populations.

Taken together, data from this method should demonstrate whether B cellsstimulated by recombinant SpA, and SAC, express a different usage of Vregions than stimulation with EBV, PMA or by cross-linking of antigenreceptors by anti-Ig.

It will be appreciated by those skilled in the art that modifications ofthe above-described steps may be desirable depending on the intended enduse of the sAg. For example, binding studies using B cells fromdifferent age populations (in particular neonatal, juvenile and elderlypopulations) will be desirable where the expected use for the sAg is asa carrier or adjuvant in a vaccine intended to enhance the immuneresponse of individuals in these populations. In addition, experimentalprotocols known to those skilled in the art to be equivalent to thoseoutlined above may be utilized to identify candidate sAg's having thecharacteristics described herein.

Further, toward constructing a specifically reactive vaccine, or toidentify for other purposes which genes and amino acid residuescorrelate with the unconventional binding sites of the sAg, molecularcloning and expression of rearranged V_(H) genes and oligonucleotidedirected site-specific mutagenesis may be used to detect which genes andresidues are critical for binding. To the same end, the cloning andexpression method described above can be used to evaluate the effects onantigen binding of CDR regions of different size and composition.

C. IDENTIFICATION OF AN ANIMAL MODEL TO ASSESS WHETHER IN VIVO EXPOSURETO B CELL sAg SKEWS THE REPRESENTATION OF IG VARIABLE REGION GENEFAMILIES.

Once a B cell sAg is identified, an animal model will be selected todemonstrate that the sAg increases the immunogenicity of bacterialand/or viral polysaccharides and/or glycoproteins in vivo.

By way of example, a murine model is appropriate for use in in vivostudies with SpA, in particular to predict the utility and function ofSpA as a superantigen in an immunization protocol performed according tothe method of the invention in humans for the following reasons. MurineV_(H) families J606 and S107 are highly homologous to members of thehuman V_(H) 3 family. Further, Fab of antibodies from these murinefamilies bind SpA, while products of other families do not. Also,antibodies from these families have been shown to be preferentially usedin formation of anti-polysaccharide antibodies on immunization withpolysaccharide--containing pathogens. Specifically, the vast majority ofhybridomas made in Balb/c mice against the capsular polysaccharide ofthe group C menigittidis use one of a small number of V_(H) genesegments from the J606 family.

Moreover, as shown in more detail in Example II, immunization of adultBalb/c mice with recombinant, endotoxin-free mod-SpA causes an increaseof circulating Ig that bind mod SpA, with recognition at or near thesame binding site on SpA which is recognized by human V_(H) 3 Fab. Theimmune response to SpA in mice can therefore be expected to besubstantially analogous to, and predictive of, the response which wouldbe achieved to immunization with the same antigen in humans. These dataindicate that the mouse would be a good animal model for in vivo studieswith the SpA sAg for use in a human and with the SpA conjugate vaccinein particular.

In contrast, data derived from binding studies using rabbit Ig andanalysis for sequence homology with human V_(H) regions of known rabbitV_(H) genes indicate that rabbit antibodies do rot interact with SpA asstrongly as human and murine antibodies. Consequently, it does notappear that the rabbit would serve as well as the mouse as an animalmodel for studies of this particular sAg. However, it will be apparentto those skilled in the art that application of the same analysis togene sequence homologies of other species with V_(H) regions in humans,combined with data concerning binding of other sAg, may indicate thatother species would serve well as animal models for use in studies ofsAg other than SpA identified according to the methods disclosed herein.

D. CONSTRUCTION OF A B CELL sAg CONJUGATE VACCINE OR COMPOSITION AND USETHEREOF IN IMMUNIZATION.

1. In vivo experimentation protocols.

Using an animal model (e.g., the murine model for use with SpA),preparations of the identified B cell sAg conjugated to a polysaccharideor glycoprotein derived from a bacterial or viral pathogen's capsule orcell wall are administered in vivo. First and generally, the sAg aloneis administered and the Ig levels of the model subjects monitored todetermine that they subsequenty retain intact B cell and T cell-mediatedimmune responses and are not immunosuppressed. Then, a series of studieswhich separately use conjugated and concomitant administrations of sAgand the polysaccharide or glycoprotein are performed using differentsizes of the polysaccharide (i.e., different-sized oligomers) andglycoprotein, and different molar ratios of polysaccharide/glycoproteinto sAg to define the optimal composition for immunogenicity, which isestablished by measurement of serum antibody titer to sAg andpolysaccharide or glycoprotein.

In addition, with particular reference to SpA, native, recombinant andmodified forms (i.e., the latter with IgG Fc binding activityeliminated) are separately used (alone and with the pathogenpolysaccharide or glycoprotein as described above) to determine whichforms are best used as the carrier or adjuvant in the inventive vaccineor composition (i.e., the composition for concomitant administration ofSpA and the polysaccharide or glycoprotein components). It is expectedthat a modified, recombinant form of SpA will be the preferred form forthis purpose.

Control animals should be immunized with conjugate vaccines using aconventional carrier protein, such as dipheria or tetanus toxoid.Efficacy of the SpA conjugate and control vaccines should be determinedin juvenile and elderly mice, which are analogous to the humanpopulations most likely to receive the vaccine.

Preferably, the animal model study would be designed so there would(statistically) be a 95% chance of detecting a 50% increase in therepresentation of the predicted V_(H) family restricted antibodies afterstimulation by the sAg/component conjugate or composition. For example,where SpA is the sAg and it is used with the murine model, a 50%increase from the baseline representation (15% ) of J606 and S107restricted antibodies would be the target for the study.

Assuming a standard deviation of 30% of mean, or unstimulated baselineof 15⁺ /-4.5%, at least 11 animals present in the control and testgroups would be required to detect the target increase (at 95%probability) would be chosen to achieve a statistical valve of 15⁺/-4.5% to 22.5%, with a P<0.05. The preferred method for determinationof V_(H) distribution and Ig binding specificity is to use a colonyhybridiaation technique with removed splenocytes reported in Kelsoe, etal. (1987), Methods in Enzymology. "Cloning of mitogen--andantigen-reactive B lymphocytes on filter paper disks: phenotypic andgenotypic analysis of B cell colonies", pp. 287-304, and Schulze, et al.(1987) J. Exp. Med. 166: 163-172, the disclosures of which areincorporated herein by this reference. In particular with respect to theuse of a murine model, it will be appreciated that baseline data on thenormal distribution of V_(H) families of BALB/c mice at different ageshave been reported and are known in the art.

Specifically preferred protocols for (1) determining the extent ofenhancement of the immune response by SpA and mod-SpA in BALB/c mice,and (2) the immunogenicity of different conjugate or preparations (orcompositions for concomitant administration) in Balb/c mice proposed foruse are:

For the first determination, using SpA, mod-SpA and a saline control, 3groups of adult 96 week old female BALB/c mice (with 12 animals in eachgroup) will be immunized so 12 mice receive SpA, 12 receive mod-SpA and12 receive saline subdermal injections. Prior to immunization, eyebleeds of 250 ul will be obtained from each animal. Then each animalwill be immunized subdermally with 0.5 ml of saline, or saline with 100mcg of SpA or saline with 50 mcg of mod-SpA. Thereafter, on weeklyintervals, eyebleeds will be obtained. The study will be continued forat least 6 weeks, or until values return to baseline.

To demonstrate the result of immunization, the blood cells can beseparated from the plasma, and analyzed separately. The blood cells willbe analyzed by flow cytometry for binding to SpA-biotin and mod-SpAcoordinately with analyses for a pan T cell marker, CD3-.sub.ε (Clone145-2c11, Pharmingen, San Diego, Calif.), or a pan B cell marker, Ly5-T(clone RA3-6B2, Pharmingen), and a B cell actlaor. These studies willdetermine whether SpA and a mod-SpA binding increases afterimmunization, and whether these cells then display the phenotype ofactivation. It will also demonstrate the kinetics of the cellularresponse.

The plasma from these bleeds will be tested by ELISA for binding tomod-SpA. In these tests the mod-SpA at 10 mcg/ml in phosphatase bufferedsaline (PBS) will be incubated on the precoat of EIA microtiter plates(COSTAR. Boston, Mass.) overnight at 4° C., then blocked for 1 hr with1% bovine serum albumin in PBS. Serial dilutions of the plasma are addedwhich are diluted in PBS for 2 hours at 15°-37° C., then washed 4 timeswith 0.05% (v/v) Tween20 in PBS, then incubated with alkalinephosphatase labeled anti-mouse IgM or alkaline phosphatase labeledanti-mouse IgG for 1 hour at 37° C., then washed 4 times, and developedwith substrate. Parallel assays will use precoats of affinity purifiedgoat anti-mouse IgM or affinity purified goat anti-mouse IgG soenrichment or depletion of the proportion of IgM of IgG Fab reactivewith mod-SpA can be detected.

Altematively, adult female Balb/c mice may be immunized either with 50mcg of recombinant SpA, or with 50 mcg of modified (non-IgG Fc binding)recombinant SpA, and a control non-immunized group. From days to weekssubsequently, the spleens will be harvested, and single cell suspensionscultured in supplemented RPMI-1640. Cultures of 200-20,000 splenocytesin 5 ml of media, with feeder cells of thymocytes depleted of B cells at1-5×10⁷ cells/ml are overplayed on Whatman 54 filter discs (8.26 cmdiameter). Colonies are grown for 5 days, and then treated with LPS,which causes a non-selective induction of RNA expression.

To detect colonies with high cytoplasmic mRNA, light microscopicevaluation of the formalin-fixed colonies after methyl-green pyroninestaining with Wright/Giemsa counterstain will be used. The colonycontaining filter is sequentially overplayed with replicate filters,which are used either for V gene, Cμ or Cγ probe hybridization, oralternatively, the filters can be immunochemically evaluated withanti-Ig, anti-idiotypic antibody or with biotinylated SpA. Serialhybridizations of each lift with three different nick-translated probescan be performed, without substantial loss of signal. Animals may alsoimmunized with bacterial proteins without using LPS in vitro with thecultured splenocytes. Therefore, as a complement to the previouslydescribed method which determines the total V_(H) distribution of Bcells present in the spleen, by omission of LPS the distribution ofV_(H) region expressed by in vivo activated B cells can be determined.

For the second determination, mod-SpA is conjugated to the purifiedpneumococcal capsular polysaccharide, Type 14, (American Tissue TypeCollection), using the conjugation method described by Peters, et al.using native, undigested polysaccharide. The conjugate is fractionatedby size and the ratio of protein to polysaccharide is determined, asdescribed previously herein.

As a starting point, the immunogenicity of preparations with 1:1 molarratios of protein to polysaccharide are tested. Thereafter, the purifiedconjugate vaccines will be administered to groups of mice as describedabove, with the groups immunized with amounts of vaccine containing 50mcg of protein equivalent. The groups will be immunized with control(saline), SpA-conjugate, mod-SpA, and purified polysaccharide alone.Although at present there is no FDA approved protein conjugate vaccinepneumococcal vaccine, experimental conjugate vaccines from commercialsources may also be compared.

Response will be measured by ELISA according to means known in the artfor anti-polysaccharide antibodies using a precoat of purifiedpolysaccharide, and the proportion of anti-polysaccharide antibodiesreactive with mod-SpA will be detected using a precoat of polysaccharideand detecting with biotinylated mod-SpA then developing withstreptavidih-alkaline phosphatase, to determine what proportion isdually reactive. Antibody levels will also be measured by standard RIA.

Subsequently, the protocol is repeated with a new set of mice todetermine the effect of using polysaccharide of different oligomersizes, changing the protein to polysaccharide ratio, and changing thedose a the immunogen. We will also test the effect of using combinationsof superaridgen and polysaccharide that are not chemically coupled.Following all protocols, splenic colony hybridization assays will beperformed to document that B cell expressing S107 and J606 VH families,the murine equivalent of the human VH3 family, are the B cell subsetsthat are modulated in size by immunizaion. This approach may also betaken to develop vaccines for other polysaccharides and glycoproteins.

For a specific comparison of the in vivo response in adult mice topneumococcal polysaccharide with and without the presence ofrecombinant, endotoxin-free mod-SpA, see Example III. As shown in theexample, the mice produced higher titers of Ig reactive with thepneumococcal polysaccharide when immunized with both the polysaccharideand modSpA than were produced on immunization with the polysaccharidealone. These data indicate that co-administration of mod-SpA and apolysaccharide can influence the resultant response to thepolysaccharide, even without chemical conjugation of the sAg to thepolysaccharide immunogen.

2. Construction of sAg vaccine or composition

The inventive method utilizes a vaccine is a chemical conjugate betweena polysaccharide or glycoprotein from a bacterial or viral source andthe sAg. Alternatively, these components may be administeredconcomitantly in a composition where in the components are not coupled.(see e.g., the comparative data produced using a pathogenic immunogenalone versus that immunogen administered concomitantly with unconjugatedmod-SpA in Example III).

Table VII lists infectious agents with pathogenicity for man whichcontain components for use in the inventive vaccine. It will beappreciated by those skilled in the art that this list is not exclusive;other human pathogens and infectious agents with pathogenicity for otherspecies may also be appropriate for inclusion in the vaccine ofcomposition preparation if they are identified as having polysaccharideor glycoprotein components which are suitable immunodominant targets.

                  TABLE 7    ______________________________________                 Diseases    ______________________________________    Bacteria    Bacillus anthracis                   anthrax    Bordetella pertussis                   whooping cough    Borrelia       a family of disease, including Lyme                   disease and relapsing fever    Campylobacter ieiuni                   gastroenteritis    Clostridium botulinum                   botulism    Corynebacterium diphtheria                   diphtheria    Escherichia coli                   many strains that produoe varied                   diseased, including diarrhea and                   urinary tract infections; some                   strains are not virulent    Haemphilus influenzae                   meningitis, epiglottitis, sinusitis,                   pneumonia, middle ear infections    Helicobacter pylori                   gastritis, duodenal ulcer diseases    Legionella pnemophila                   Legionnaires' disease    listeria monocytogenes                   meningitis, sepsis    Mycobacterium lepraie                   leprosy    Mycobacterium tuberculosis                   tuberculosis    Neisseria gonnorrhoeae                   gonorrhea    Neissena meningitidis                   sepsis, meningitis    Norcadia ateroides                   lesions of the respiratory tract    Pseudomonas aeruginosa                   infections of compromised hosts,                   nonsocomial infections    Rickettsia     Rocky Mountain spotted fever, typhus                   (rare)    Salmonella     typhoid fever, enteric fever,                   gastroenteritis    Shigella       dysentery    Staphylococcus aureus                   impetigo, boils, soft tissue and                   systemic infection, osteomyelitis,                   toxic shock syndrome    Streptococcus pyogenes                   pneumonia, sinusitis, meningitis, otitis                   media    Streptococcus pyogenes                   rheumatic fever, soft tissue infection,                   pharyngitis    Treponema pallidum                   syphilis    Vibrio cholerae                   cholera    Yersinia pestis                   bubonic plague    Yersinia enterocolitica                   mesenteric lymphadenitis    and pseudotuberculosis    Viruses    cytomegalovirus                   mononucleosis    dengue virus   dengue fever    Epstein-Barr virus                   mononucleosis, Burkitts lymphoma    hepatitis B virus                   hepatitis    herpes simplex type 1                   encephaitis, stomatitis    herpes simplex type 2                   genital lesions    HIV            acquired immunodeficiency syndrome    HTLV-1         T cell leukemia    influenza virus                   upper respiratory tract infection,                   pneumonia    measles virus  measles    papillomavirus warts, associated with later-developing                   cervical cancer    pneumovirus    respiratory infection    polyomavirus   malignant tumors in animals    reoviridae     diarrhea, Colorado tick fever    rhabdoviruses  rabies    rhinovirus     common cold    ribvirus       rubella    varicella-zoster                   chickenpox, herpes zoster (shingles)    ______________________________________

In particular, microbial products which stimulate the same V_(H) familyrestricted antibodies which are preferentially stimulated by the sAg areexpected to be efficaciously treated with a vaccine or compositionutilizing the sAg as a carrier or adjuvant. With respect to SpA,microorganisms with components which stimulate V_(H) 3 restrictedantibodies are now known to include the encapsulated bacteriaHaemophilus influenzae Type b (Hib) (see, Silverman and Lucas (1991) J.Clin. Invest. 88:911-920 the disclosure of which regarding theexperimental protocols used to identify Hib as stimulating V_(H) 3restricted antibodies is incorporated herein!) and Streptococcuspneumoniae.

With respect to the latter, pneumococcal polysaccharides also stimulateproduction of V_(H) 3 restricted antibodies as shown in the experimentdescribed below.

As shown by the data in FIGS. 5-7, 20 healthy human adults wereimmunized with the polyvalent PNEUMOVAX vaccine (Merck Sharpe and Dohme)and pre- and post-immunization levels to antibodies to each of the 24different purified polysaccharides in the vaccine were measured.Responders were then selected, who had over three fold increase inpost-immunization IgG antibody responses, and antibodies to a particularcapsular type were purified by direct precipitation, following exactlythe method described in Silverman and Lucas (J. Clin Invest (1991)88:911-920).

Following this protocol, the purified antibody antigen complexes wereseparated by PAGE electrophoresis under reducing conditions, and thevariable region composition of the antibodies was assessed by subjectingreplicate immunoblots to analysis with a series of serologic reagentsthat are each specific for particular human immunoglobulin variableregion families. Thereby, it was established that several antibodyresponses examined in detail, C-polysaccharide, Type 14, and Type 19,exclusivety used or were highly biased toward the usage of the V_(H) 3family, while many V_(L) families were used within a particular responsein an individual.

Referring to Tables VIII-X, a particular subject is listed at left, thenthe strength of reactivity of his polysaccharide specific antibodieswith each of the serologic reagents is listed beneath the name of thereagent. V_(H) 1, V_(H) 4 and V_(H) 3 represent reagents that identifymost or all members of their respective families. Subsets within theV_(H) 3 family are identified by the V_(H) 3-V_(H) 2a and V_(H) 3-FR3aantibodies that identify different component on one type of V_(H) 3 Hchain, and V_(H) 3-HV2b which identifies another distinct, separatesmall subset of V_(H) 3 H chains. These reagents and their specificityare described in the J. Clin Invest paper, but the significant findingis that similar H chains are used in anti-Hib PS (polysaccharide) andanti-pneumococcal responses. Antibodies that contain H chains from theseV_(H) 3 subsets are shown to react with the alternative binding of SpA.In contrast, there is little or no restriction of V_(L) usage byanti-pneumococcal antibodies.

It is expected based on success with conventional vaccines that theinventive vaccine will also be particularly useful in enhancing immuneresponse to Neiserria meningitidis.

Once an optimal size for the pathogen component as well as the bestmolar ratio of component to sAg, has been determined according to theabove-described methods, construction of a conjugate vaccine willgenerally be performed according to method reported for preparation ofconventional polysaccharide vaccines by Peters, et al. (1991) 30:267-274(discussion at anti-Hib, anti-meningitidis and anti-pneumococcalconjugate vaccines). Other references of interest in this regard areTsai, et al. (1989) J. Biol. Stand. 17:249-258; Verhuel, et al. (1989)Infection and Immunity 57:1078-1083, and Allison, et al. (1991) Molec.Immun. 28:279-284 (all of these references are incorporated herein).

In particular with respect to SpA, the preferred form of SpA is expectedto be a highly purified recombinant form available commercially fromCalbiochem, which is sold under the trade name ULTRAPURE (rSpA). It isexpected that the most efficacious form of SpA for use in the vaccinewill be one which has been hyperiodinated to eliminate Fc bindingactivity (i.e., mod-SpA). Hyperiodnation is performed as follows: 1 mgof SpA is dissolved in 350 ul of 5× borate buffered saline (BBS; ph8.2). Under a fume hood, on ice, 600 ul of 0.02M iodine monochloride isadded dropwise with agitation. This solution is incubated for 30 minutesat 4° C., then the mixture is dialyzed against 1× BBS or PBS, with threechanges of buffer over 48 hours at 4° C.

Other forms of SpA which may be used are the purified native form orpurified, modified (hyperiodinated) form. The recombinant form ispreferred because it is expressed in a E.coli strain which contains theProtein A gene from a S. aureus Cowan I strain (SAC). As a result, rSpA(in particular the ULTRAPURE SpA product) is free of staphylococcalenterotoxins and hemolysins. rSpA has also been shown to achieve ahigher purity level than the purified, native S. aureus produced ProteinA. rSpA from E. coli also has higher solubility and expression ofprotein levels than S. aureus derived Protein A; their binding activityis comparable to IgG.

Further, it should be noted with respect to SpA that it contains fourIgG F_(c) binding activity (regions D,A, B and C). ft is known that SpAhas at least two Fab binding sites, but they have not as yet beenlocalized. However, due to the need to cross-link Fab for superantigen Bcell stimulation, at least two Fab binding sites on the vaccine sAg areexpected to be required and may be present in each region, thus allowingfor use of a D, A, B or C fragment in the vaccine rather than whole SpA.

Several variables come into play in preparing the conjugate vaccine(which to some extent are considerations for the unconjugated vaccine aswell), including polysaccharide/sAg protein or glycoprotein/sAg proteinratio, solubility use of spacers, monosaccharide composition and, incertain uses the degree cross-linking in presence of an additionaladjuvant. Generally, preparation of test vaccines which vary as againsta control in these factors (including the use of oligo-saccharides inlieu of polysaccharides) and immunization of different test and controlgroups win, without undue experimentation, yield sufficient informationto determine an optimal structure for the vaccine.

Suitable experimental protocols are reported in Verhuel, et al. supra,and Peters, et al. suora (regarding polysaccharide/protein ratios, see,in particular, Verhuel et al. at Table I therein for reference). It isexpected that the sAg/component vaccines produced and used according tothis invention which incorporate poly- or oligosaccharide componentswill use the soluble carbodiimide conjugation method, preceded byperiodate oxidation (and, for mod-SpA, hyperiodination) of thepolysaccharide prior to linkage with a 6-aminohexanoic acid spacer bycyanogen bromide (see Peters, supra; and, Chu, et al. (1983) Infect.Immunol. 40:245-256 incorporated herein by this reference!).Alternatively, the polysaccharide can be activated with cyanogen bromidethen reacted with adipic dihydrazide to make a polysaccharide hydrazidederivative prior to carbodiimide conjugation. Other spacers have alsobeen described by Marburg, et al. (1986) J. Am. Chem. Soc.108:5282-5287.

Generally, it can be expected that oligosaccharides will be moreimmunogenic than polysaccharides (although Peters, et al. reported thatthe opposite was true in a type 4 S.pnuemoniae conjugate vaccine).Further, the use of spacer molecules is expected to be preferred.However, based on the expected enhancement of the immune response by thesAg, use of an additional adjuvant (such as the polysacoharide totiterpenoid quillaic acid conjugate) should not be necessary ordesirable.

It is not entirely clear based on work to date that conjugation of thesAg and component will be required to stimulate the desired immuneresponse. These may, therefore, be tested and administered in anunconjugated composition of purified sAg/component supematants. In thisrespect, it is expected that it may be desirable to maintain thepercentage of unconjugated component in the composition to less than 10%to avoid inducing immunosuppression. Alternatively, the immune responsemay be enhanced by administration of equally low dosages of unconjugatedcomponent prior to administration of the conjugated vaccine.

Administration may be subcutaneous, intramuscular, subdermal,intravenous, intraperitoneal or intradermal; the former is preferred.Resulting circulating antibody levels will be verified by immunoassaymeans well-known in the art; i.e., modified radioimmunoassay orcompetition ELISA (see e.g., method reported for use with conventionalpneumococcal vaccines in Peters, et al. 1991, J. Immunol. 146:4308-4314;see also, the ELISA protocols described in Example II and III below).

For construction of conjugates having a glycoprotein component,different chemical conjugation means are used in methods similar tothose described above. Carboniimide, glutaraldehyde, bis-diazotizedbenzidine or m-maleimido-benzoyl-N-hydroxysuccinimide ester may be usedas cross-linkers to perform the conjugation. Specific suitable couplingmethods for conjugation of peptides (including glycoproteins) to carrierproteins are described in Harlow et al. "Antibodies: a laboratorymanual", Cold Spring Harbor Laboratory (1988), Ch. 5, 78-87.

It should be noted that not all of the above methods have been approvedfor use in vaccines intended for administration to humans. As a result,while all are suitable for use in testing as described above, the finalproduction conjugation method may have to be modified to adapt tothen-currently approved methods for use in humans.

The invention having been fully described, examples illustrating itspractice are provided below. However, it will be understood that theinvention, defined by the appended claims, is not limited by theexamples and that modifications may be made to the invention asdescribed therein and in the specification without departing from itsspirit or scope.

EXAMPLE I IDENTIFICATION OF THE V_(H) 3 RESTRICTED IG (Fab) BINDING SITEON SpA

To determine whether the D domain of SpA will bind Ig Fab (of, inparticular, V_(H) 3 restricted Ig), the D domain has been cloned andexpressed in vitro. This was performed by use of the polymerase chainreaction, using two oligonucleotide primers that flank the D domain.

1. Primer Construction.

The upstream primer, DomD 5' (SEQ.ID.No. 49), includes the DNA sequenceof a three codon insertion unique to SpA domain D (and E) so that the Ddomain would be selectively amplified. The other primer, DomDAS 3'(SEQ.ID.No. 50), includes an antisense sequence that is about 232nucleotides away from the first primer. Together, in the conditionsindicated below, these primers amplified an about 232 base pair fragmentof DNA.

These primers also include some differences from the natural SpAsequence which create new potential DNA restriction sites. This band ofDNA was then purified, and the DNA was digested at specific sites withthe restriction enzymes BamHI and BgIII, that had been engineered intothe oligonucieotide primers. The DNA fragment was then purified again byagarose gel electrophoresis, and it was ligated into a property preparedcloning site into a modified pUC19 plasmid, which allowed directionalcloning of the fragment into a position in which it was grafted at the3' end of a ompA leader sequence. This gene is also under the control ofan upstream Lac operon. The plasmid includes the ampicillin resistancegene that enables selection of plasmid containing bacteria by antibioticselection. Therefore, after introduction into a compatible E. Colistrain, and selection with ampicillin or carbenicillin, expression ofthe D domain can be induced by the standard IPTG method.

For the PCR amplification, the plasmid, pSpA8, which contains the SpAgenome was obtained from the American Type Culture Collection inRockville, Md. and used as a template. The following reaction conditionswere employed:

94° C. for 1 minute, 80° C. for 1 minute (and the Taq polymerase isadded), then

30 cycles of:

93° C. for 1 min

46° C. for 2 min

72° C. for 2 min, then

72° C. for 10 minutes.

These reactions were performed in an "ERICOMP" thermocycler (San Diego,Calif.) in a 100 ul final volume, containing each primer in a finalconcentration of 0.01 pM/ul, MgC12 at 1.0 mM, 20Q microM of each of thedNTP, 1 ng of the template DNA and a total of 5 units of Taq polymerase.As controls, identical tubes that lacked one of the primers, and a tubethat lacked only the template DNA were treated under identicalconditions. These control tubes failed to produce DNA of the predictedsize, as predicted.

To analyze Ig binding capacity the bacterial extracts that include theDomain D product were subjected to size separation of the componentproteins under reducing conditions (1% 2-mercaptoethanol) on 15%polyacrylamide gel electrophoresis. For comparison, extracts of bacteriathat did not contain the plasmid were prepared in the same manner(negative control), SpA from a commercial source (positive control), andMW markers were run on adjacent lanes to the Domain D containing extractThey were then electrotransferred onto a "IMMOBILON" pR membrane(Millipore, Bedford, Mass.). The membranes were then subjected toimmunoblotting with different Ig preparations. These studiesdemonstrated that Domain D binds to a component of purified polydonalIgM from adult donors, and also that it binds to monoclonal VH3 IgM.

The predicted protein sequence for the portion of the D domain of SpAthat binds Fab is the following 61 amino acids (SEQ.ID.No. 51):

ADAQQNNFNKDQQSAFYEILNMPLNEAQRNGEFIOSTKDDPSOSTNVLGEAKKLNESQAPK

EXAMPLE II RESPONSE TO SpA IMMUNIZATION IN A MURINE MODEL AND UTILITY OFTHE MODEL FOR PREDICTION OF THE RESPONSE TO SpA IN HUMANS

A. In vivo Production of Murine Ig Against Mod-SpA

A sample of recombinant SpA free of E.coli endotoxins was purchased froma commercial source (Repligen, Cambridge, Mass.) and the SpA wasmodified by hyperiodination to remove Fc binding as described elsewhereabove. Balb/c mice (in groups of three), were immunized subcutaneouslyon their backs with 100 mcg of the mod-SpA in an alum emulsion in 0.5 mlof normal saline at day 0, and 50 mcg.ml at week 8. A control groupreceived sham (i.e., saline only) injections. Blood samples were takenperiodically and enzyme-linked immunoabsorbent assays (ELISA's)performed on the samples as follows.

Experimentally naive adult female Balb/c mice were immunized with 100mcg of endotoxin-free mod-SpA at day 0, and with 50 mcg/ml at week 8.Blood samples were obtained periodically, and immune assay performed asfollows. Duplicate microtiter wells were precoated with mod-SpA at 5mcg/ml in phosphate buffered saline, pH 7.4 (PBS) overnight at 4° C.Plates were then blocked with 1% bovine serum albumin (BSA) in PBS for 1hr at RT. In duplicate, samples diluted 1:5000 in BSA-PBS were incubatedon plates for 2 hr at 37° C. Plates were then washed and then incubatedwith either affinity purified goat anti-mouse IgG-horse radishperoxidase, or affinity purified goat anti-mouse IgM-horse radishperoxidase, for 1 hour at room temperature. Plates were then washed,then developed with substrate and optical density was read at 405 A withoptical density of 490 A subtracted. The background reading of wellswithout mouse serum samples was 0.050.

Fc binding activity was undetectable in an ELISA that was sensitiveenough to detect less than 3% of the original activity, but Ig Fabbinding capacity was preserved. Also, as determined by flow cytometrythe total number of B cells in these mice at least doubled, but theproportion of B lymphocytes that bound the mod-SpA did not change.

However, assays of Ig binding of mod-SpA indicated that the animalsdeveloped an immune response to immunization, which was boosted aftersecondary immunization with mod-SpA These mice were shown to have anincrease in the titers of mod-SpA binding Ig within 24 weeks afterprimary immunization (FIG. 5). Ig binding levels increased 1.5 to about4 fold with peaks between 4 and 7 weeks and then declined. Secondaryimmunization with 50 mcg of mod-SpA administered by the same routeinduced more rapid and greater increases with greater increases in theIgG than the IgM binding of mod-SpA. Overall, most of the response wasIgG. with lesser increases in IgM mod-SpA binding.

Because the mice immunized with mod-SpA continue to have increasedlevels of Ig that bind mod-SpA for 14 weeks after secondaryimmunization, it can be concluded that, unlike the effect of exposure ofcertain T cell superantigens to T lymphocytes, immunization with mod-SpAdoes not result in subsequent B cell unresponsiveness to secondaryexposure to mod-SpA

B. Inhibition of SpA Binding of Anti-SpA Murine Ig by a Human MonoclonalAntibody.

Inhibition studies documented that immunization caused an increase inthe binding of murine Ig against a SpA site that is also bound bymonoclonal human V_(H) 3 IgM (FIG. 6).

The experiment was performed as indicated above, except that afterblocking of each plate, one half of each plate was preincubated withaffinity purified SpA3-08 Fab at 5 mcg/ml in BSA-PBS while the otherpart of each plate had only 1% BSA-PBS, for 2 hr at 37° C. Serialdilution of a post-immunization mouse sera, in triplicate, were thenadded to both sides of the plate and incubated for 2 hr at 37° C. Plateswere then washed and developed as above, except using affinity purifiedgoat anti-mouse IgG and IgM-horse radish peroxidase. Preincubation ofthe mod-SpA with the SpA3-08 Fab, caused about a 2/3 reduction of themurine Ig binding to mod-SpA in the post-immunization sample.

In one mouse examined, an inhibition study established that 2/3 of themod-SpA binding in the secondary response to mod-SpA could be blocked bypreincubation of the mod-SpA with a human monoclonal IgG Fab, termedSpA3-08. This confirms that SpA immunization of adult Balb/c mice causesan increase of circulating V_(H) 3 restricted Ig that bind mod-SpA,which response is predominately directed at (or near) the human VH3 Fabbinding site on SpA.

EXAMPLE III CO-IMMUNIZATION OF PNEUMOCOCCAL POLYSACCHARIDE AND mod-SpAINFLUENCES THE RESPONSE TO PNEUMOCOCCAL POLYSACCHARIDE

Groups of four experimentally naive adult female mice weresubcutaneously immunized with 0.5 ml of normal saline emulsified in alumwhich contained either a 1:50 dilution of "PNEUMOVAX" pneumococcalvaccine (Merck Sharpe and Dohme, West Point, Pa.) or 1:50 dilution of"PNEUMOVAX" with 50 mcg of mod-SpA (endotoxin-free). Pre-immunizationresponses were then compared with 6 weeks postimmunizaton samples.

To this end, ELISA's were performed as described in Example II exceptthat precoats utilized purified polysaccharide 14 (Merck, Shaerpe andDohme) at 20 mcg/ml in phosphate buffered saline (PBS) overnight at 4°C. Sera were diluted 1:100 in BSA-PBS and preincubated for 2 hours at37° C. with pneumococcal cell wall, then incubated on the plates for 2hours at 37° C. Plates were then washed, then incubated withmod-SpA-biotin for 1 hour at room temperature, then washed and developedwith avidin-horseradish peroxidase.

All animals had increased titers to type specific capsular pneumococcalpolysaccharide 14. The levels were not significantly different inimmunoassays of sera using a precoat of polysaccharide 14 and adetecting agent of affinity purified goat anti-mouse IgG and IgMhorseradish peroxidase (data not shown). However, when the studies wererepeated substituting labelled mod-SpA as the detecting agent, the levelof mouse Ig reactive with both mod-SpA and polysaccharide 14 wasdifferent between the groups (FIG. 7). A higher level of dually reactivemouse Ig was present in each mouse that received both mod-SpA and"PNEUMOVAX" compared to every mouse that received "PNEUMOVAX" alone.These data indicate that co-administration of mod-SpA and apolysaccharide, even without chemical conjugation, can influence theresultant response to the polysaccharide.

SUMMARY OF SEQUENCES

SEQUENCE ID NO's: 1-11 are the amino acid sequences for intact IgM andIgG antibodies with significant mod-SpA binding activity which are shownin Table II.A.

SEQUENCE ID NO's: 12-18 are amino acid sequences for monoclonalantibodies (non-VH3 origin) which are devoid of SpA binding activity andwhich are shown in Table II.A.

SEQUENCE ID NO's: 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40 areamino acid sequences for monoclonal Fab which were isolated from acombinatorial library based on SpA binding capacity and which are shownin Table II.A.

SEQUENCE ID NO's: 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 42,43, 44, 45, 46, 47 and 48 are nucleotide sequences and correspondingamino acid sequences for the variable heavy in regions of SpA bindersfrom combinatorial libraries which are depicted in Table II.B. SEQUENCEID NO: 42, 44, 46 and 48 correspond to the nucleotides shown in SEQUENCEID NO: 41, 43, 45 and 47.

SEQUENCE ID NO's: 49-50 are amino acid sequences of primers used toamplify Domain D of SpA.

SEQUENCE ID NO: 51 is the amino acid sequence of Domain D of SpA.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 51    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 125 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (vii) IMMEDIATE SOURCE:    (B) CLONE: 18/2    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..125    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    GluValGlnLeuLeuGluSerGlyGlyGlyLeuValGlnProGlyGly    151015    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerTyr    202530    AlaMetSerTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal    354045    SerAlaIleSerGlySerGlyGlySerThrTyrTyrAlaAspSerVal    505560    LysGlyArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyr    65707580    LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCys    859095    ThrLysGlyGlnValLeuTyrTyrGlySerGlySerTyrHisTrpPhe    100105110    AspProTrpGlyGlnGlyThrLeuValThrValSerSer    115120125    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 116 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (vii) IMMEDIATE SOURCE:    (B) CLONE: ED8.4    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..116    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    GluValGlnLeuLeuGluSerGlyGlyGlyLeuValGlnProGlyGly    151015    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerTyr    202530    AlaMetSerTrpValLeuGlnProProGlyLysGlyLeuGluTrpVal    354045    SerAlaIleSerGlySerGlyGlyArgThrTyrTyrAlaAspSerVal    505560    LysGlyArgPheThrIleSerArgGluAsnSerLysAsnThrLeuTyr    65707580    ValGlnMetAsnSerLeuArgValGluAspThrAlaValTyrTyrCys    859095    AlaLysGlyTyrGlyMetAspValTrpTrpGlyGlnGlyThrLeuVal    100105110    ThrValSerSer    115    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 109 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE:    (vii) IMMEDIATE SOURCE:    (B) CLONE: Huab14-3    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..109    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    GluValGlnLeuValGluSerGlyGlyGlyLeuValGlnProGlyGly    151015    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerThrTyr    202530    ArgMetHisTrpValArgGlnAlaProGlyLysGlyLeuValTrpVal    354045    SerArgIleAsnSerAspGlySerSerThrAspTyrAlaAspSerVal    505560    GluGlyArgPheThrIleSerArgAspAsnAlaLysAsnThrLeuTyr    65707580    LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrPhe    859095    AspSerTrpGlyGlnGlyThrLeuValThrValSerSer    100105    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 108 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (vii) IMMEDIATE SOURCE:    (B) CLONE: SFL    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..108    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    GluValGlnLeuValGluSerGlyGlyGlyLeuValGlnProGlyGly    151015    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheThrXaaTyr    202530    XaaMetSerTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal    354045    AlaPheIleTyrArgSerGlyIleAsnLysTyrTyrAlaAspSerVal    505560    AsnGlyArgPheThrIleSerArgAspAsnXaaLysAsnThrLeuXaa    65707580    XaaXaaMetAsnSerLeuArgAlaGluAspThrAlaLeuTyrTyrCys    859095    AlaLysAspAlaGlyLeuLysValGluLysSerVal    100105    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 125 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (vii) IMMEDIATE SOURCE:    (B) CLONE: KIM    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..125    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    GluValGlnLeuValGluSerGlyGlyGlyValValGlnProGlySer    151015    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerPhe    202530    AlaMetHisTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal    354045    AlaValMetSerTyrSerGlyAspAsnLysTyrTyrValAspSerVal    505560    LysGlyArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyr    65707580    LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaLeuTyrTyrCys    859095    AlaLysLeuSerThrAlaAlaSerGlyPheThrPheAspThrTyrGly    100105110    MetAspTrpGlyGlnThrThrLeuValThrValSerSer    115120125    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 125 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (vii) IMMEDIATE SOURCE:    (B) CLONE: RIV    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..125    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    GluValGlnLeuValGluSerGlyGlyGlyValValGlnProGlySer    151015    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerPhe    202530    AlaMetHisTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal    354045    AlaValMetSerTyrSerGlyAspAsnLysTyrTyrValAspSerVal    505560    LysGlyArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyr    65707580    LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaLeuTyrTyrCys    859095    AlaLysLeuSerThrAlaAlaSerGlyPheThrPheAspThrTyrGly    100105110    MetAspTrpGlyGlnThrThrLeuValThrValSerSer    115120125    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 120 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (vii) IMMEDIATE SOURCE:    (B) CLONE: LAY    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..120    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    AlaValGlnLeuLeuGluSerGlyGlyGlyLeuValGlnProGlyGly    151015    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerAlaSer    202530    AlaMetSerTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal    354045    AlaTrpLysTyrGluAsnGlyAsnAspLysHisTyrAlaAspSerVal    505560    AsnGlyArgPheThrIleSerArgAsnAspSerLysAsnThrLeuTyr    65707580    LeuGlnMetAsnGlyLeuGlnAlaGluValSerAlaIleTyrTyrCys    859095    AlaArgAspAlaGlyProTyrValSerProThrPhePheAlaHisTrp    100105110    GlyGlnGlyThrLeuValThrVal    115120    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 122 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (vii) IMMEDIATE SOURCE:    (B) CLONE: POM    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..122    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    GluValGlnLeuLeuGluSerGlyGlyGlyLeuValGlnProGlyGly    151015    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerSer    202530    AlaMetSerTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal    354045    AlaTrpLysTyrGluAsnGlyAsnAspLysHisTyrAlaAspSerVal    505560    AsnGlyArgPheThrIleSerArgAsnAspSerLysAsnThrLeuTyr    65707580    LeuLeuMetAsnSerLeuGlnAlaGluAspThrAlaLeuTyrTyrCys    859095    AlaArgAspAlaGlyProTyrValSerProThrPhePheAlaHisTyr    100105110    GlyGlnGlyThrLeuValThrValSerSer    115120    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 125 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (vii) IMMEDIATE SOURCE:    (B) CLONE: Huab2-3    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..125    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    GluValGlnLeuValGluSerGlyGlyGlyLeuValGlnProGlyGly    151015    SerLeuLysLeuSerCysAlaAlaSerGlyPheThrPheSerAlaSer    202530    AlaValHisTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal    354045    GlyArgIleArgSerLysAlaAsnSerTyrAlaThrAlaTyrAlaAla    505560    SerValLysGlyArgPheThrIleSerArgAspAsnSerLysAsnThr    65707580    AlaTyrLeuGlnMetAsnSerLeuLysThrGluAspThrAlaValTyr    859095    TyrCysThrGlyHisProLeuTyrTyrValThrThrProHisTrpPhe    100105110    AspProTrpGlyGlnGlyThrLeuValThrValSerSer    115120125    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 116 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (vii) IMMEDIATE SOURCE:    (B) CLONE: SB5/D6    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..116    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    GluValGlnLeuValGluSerGlyGlyGlyLeuValLysProGlyGly    151015    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerAsnAla    202530    TrpMetAsnTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal    354045    GlyArgIleLysThrLysThrAspGlyGlyThrThrAspTyrAlaAla    505560    ProValLysGlyArgPheThrIleSerArgAsnAspSerLysAsnThr    65707580    LeuTyrLeuGlnMetAsnSerLeuLysThrGluAspThrAlaValTyr    859095    TyrCysThrThrGlyGlyGlyValGlyTrpGlyGlnGlyThrLeuVal    100105110    ThrValSerSer    115    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 119 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (vii) IMMEDIATE SOURCE:    (B) CLONE: 4B4    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..119    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    GluValGlnLeuValGluSerGlyGlyGlyLeuValLysProGlyGly    151015    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerAsnAla    202530    TrpMetSerTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal    354045    GlyArgIleLysSerLysThrAspGlyGlyThrThrAspTyrAlaAla    505560    ProValLysGlyArgPheThrIleSerArgAspAspSerLysAsnThr    65707580    LeuTyrLeuGlnMetAsnSerLeuLysThrGluAspThrAlaValTyr    859095    TyrCysThrThrAspSerLeuProProHisArgValTrpGlyGlnGly    100105110    ThrLeuValThrValSerSer    115    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 120 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (vii) IMMEDIATE SOURCE:    (B) CLONE: BOR    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..120    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    ValGlnLeuValGlnSerGlyAlaGluValLysLysProGlySerSer    151015    ValLysValThrCysLysAlaSerGlyAspThrPheSerSerSerAla    202530    IleSerTrpValArgGlnAlaProGlyGlnGlyLeuGluTrpMetGly    354045    GlyIleIleProIlePheGlyThrProAsnTyrAlaGlnLysPheGln    505560    GlyArgValThrIleThrThrAspGluSerThrSerThrAlaTyrMet    65707580    GluValSerSerLeuArgSerGluAspThrAlaLeuTyrTyrCysAla    859095    ArgGluGlyArgArgMetAlaIleAsnProPheAspTyrTrpGlyGln    100105110    GlyThrLeuValThrValSerSer    115120    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 120 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (vii) IMMEDIATE SOURCE:    (B) CLONE: KAS    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..120    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    ValHisLeuValGlnSerGlyAlaGluValLysLysProGlySerSer    151015    ValLysValSerCysLysAlaSerGlyGlyThrPheSerSerTyrAla    202530    IleSerTrpValArgGlnAlaProGlyGlnGlyLeuGluTrpMetGly    354045    GlyIleIleProIlePheGlyGlnAlaAsnTyrAlaGlnLysPheGln    505560    GlyArgValThrIleThrAlaAspGluSerThrAsnThrAlaTyrMet    65707580    GluLeuArgSerLeuArgSerAspAspThrAlaMetTyrTyrCysAla    859095    LysGluGlyTyrGlyAspTyrGlyArgProPheAspPheTrpGlyGln    100105110    GlyThrLeuValThrValSerSer    115120    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 121 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (vii) IMMEDIATE SOURCE:    (B) CLONE: SIE    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..121    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..121    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    ValGlnLeuValGlnSerGlyAlaGluValLysLysProGlySerSer    151015    ValArgValThrCysLysThrSerGlyGlyThrPheSerGlyTyrThr    202530    IleSerTrpValArgGlnAlaProGlyArgGlyLeuGluTrpValGly    354045    SerProAlaLysTrpThrAspProPheGlnGlyValTyrIleLysTrp    505560    GluArgValThrValSerLeuLysProSerPheAsnGlnAlaTyrMet    65707580    GluLeuValAsnLeuPheAsnGluAspGlyAlaValTyrTyrCysAla    859095    ArgGluTrpLysGlyGlnValAsnValAsnProPheAspTyrTrpGly    100105110    GlnGlyValLeuValThrValSerSer    115120    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 128 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (vii) IMMEDIATE SOURCE:    (B) CLONE: CESS    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..128    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    GlnValAsnLeuArgGluSerGlyProAlaLeuValLysAlaThrHis    151015    ThrLeuThrLeuThrCysThrPheSerGlyLeuSerValAsnThrArg    202530    GlyMetSerValSerTrpIleArgGlnProProGlyLysAlaLeuGlu    354045    TrpLeuAlaArgIleAspTrpAspAspAspLysTyrTyrGlyThrSer    505560    LeuGluThrArgLeuThrIleSerLysAspThrSerLysAsnGlnVal    65707580    ValLeuLysValThrAsnMetAspProAlaAspThrAlaThrTyrTyr    859095    CysAlaArgMetGlnValThrMetValArgGluValMetIleThrSer    100105110    AsnAlaPheAspIleTrpGlyGlnGlyThrMetValThrValSerSer    115120125    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 119 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (vii) IMMEDIATE SOURCE:    (B) CLONE: LES    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..119    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    GlnValGlnLeuGlnGlnTrpGlyAlaGlyLeuLeuLysProSerGlu    151015    ThrLeuSerLeuThrCysAlaValTyrGlyGlyProPheSerGlyTyr    202530    TyrTrpSerTrpValArgGlnProProGlyLysGlyProGluTrpIle    354045    GlyGluIleAsnHisSerGlyArgThrThrTyrAsnProSerLeuThr    505560    SerArgValThrMetSerLeuAspThrSerLysAsnGlnPheSerLeu    65707580    LysLeuThrSerValThrAlaAlaAspThrAlaValTyrTyrCysAla    859095    ArgGlyProCysGluAlaTyrCysThrAspAspAlaProGlnAlaTyr    100105110    PheGlnHisTrpGlyGlnGly    115    (2) INFORMATION FOR SEQ ID NO:17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 98 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (vii) IMMEDIATE SOURCE:    (B) CLONE: A224    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..98    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    GluValGlnLeuValGlnSerGlyAlaGluValLysLysProGlyGlu    151015    SerLeuLysIleSerCysLysThrSerGlyTyrSerPheThrAsnTyr    202530    TrpIleGlyTrpValArgGlnMetProGlyLysGlyLeuGluTrpMet    354045    GlyLeuIleTyrProGlyAspSerGlyThrArgTyrSerProSerPhe    505560    LysGlyGlnValThrIleSerAlaAspLysSerIleSerThrAlaTyr    65707580    LeuGlnTrpSerSerLeuArgAlaSerAspThrAlaMetTyrTyrCys    859095    AlaArg    (2) INFORMATION FOR SEQ ID NO:18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 120 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (vii) IMMEDIATE SOURCE:    (B) CLONE: L16    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..120    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    GlnValGlnLeuGlnGlnSerGlyProGlyLeuValLysProSerGln    151015    ThrLeuSerLeuThrCysAlaIleSerGlyAspSerValSerSerAsn    202530    SerAlaAlaTrpAsnTrpIleArgGlnSerProSerArgGlyLeuGlu    354045    TrpLeuGlyArgThrTyrTyrArgSerLysTrpTyrAsnAspTyrAla    505560    ValSerValLysSerArgIleThrIleAsnProAspThrSerLysAsn    65707580    GlnPheSerLeuGlnLeuAsnSerValThrProGluAspThrAlaVal    859095    TyrTyrCysAlaArgGluLeuGlyAspAlaPheAspIleTrpGlyGln    100105110    GlyThrMetValThrValSerSer    115120    (2) INFORMATION FOR SEQ ID NO:19:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 360 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (vii) IMMEDIATE SOURCE:    (B) CLONE: SpA3-02    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..360    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    CAGGTGAAACTGCTCGAGTCTGGGGGAGGATTGGTACAGCCTGGGGGG48    GlnValLysLeuLeuGluSerGlyGlyGlyLeuValGlnProGlyGly    151015    TCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCCAT96    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerHis    202530    GCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGCCTGGAGTGGGTC144    AlaMetSerTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal    354045    TCAGATATTAGTGCCAGTGGTGGTAGCACATATTATGCAGACTCCGTG192    SerAspIleSerAlaSerGlyGlySerThrTyrTyrAlaAspSerVal    505560    AAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTAT240    LysGlyArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyr    65707580    TTGCAAATGAACAGCCTGAGAGCCGAAGACACGGCCTTATATTACTGT288    LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaLeuTyrTyrCys    859095    GCGTCCAACGGCGCGGGATGGGGGCTACCTTCCCTTGACTACTGGGGC336    AlaSerAsnGlyAlaGlyTrpGlyLeuProSerLeuAspTyrTrpGly    100105110    CAGGGAACCCTGGTCACCGTCTCC360    GlnGlyThrLeuValThrValSer    115120    (2) INFORMATION FOR SEQ ID NO:20:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 120 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    GlnValLysLeuLeuGluSerGlyGlyGlyLeuValGlnProGlyGly    151015    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerHis    202530    AlaMetSerTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal    354045    SerAspIleSerAlaSerGlyGlySerThrTyrTyrAlaAspSerVal    505560    LysGlyArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyr    65707580    LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaLeuTyrTyrCys    859095    AlaSerAsnGlyAlaGlyTrpGlyLeuProSerLeuAspTyrTrpGly    100105110    GlnGlyThrLeuValThrValSer    115120    (2) INFORMATION FOR SEQ ID NO:21:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 360 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (vii) IMMEDIATE SOURCE:    (B) CLONE: SpA3-08    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..360    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    CAGGTGAAACTGCTCGAGTCTGGGGGAGGATTGGTACAGCCTGGGGGG48    GlnValLysLeuLeuGluSerGlyGlyGlyLeuValGlnProGlyGly    151015    TCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCCAT96    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerHis    202530    GCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGCCTGGAGTGGGTC144    AlaMetSerTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal    354045    TCAGATATTAGTGCCAGTGGTGGTAGCACATATTATGCAGACTCCGTG192    SerAspIleSerAlaSerGlyGlySerThrTyrTyrAlaAspSerVal    505560    AAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTAT240    LysGlyArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyr    65707580    TTGCAAATGAACAGCCTGAGAGCCGAAGACACGGCCTTATATTACTGT288    LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaLeuTyrTyrCys    859095    GCGTCCAGCGGCGCGGGATGGGGGCTACCTTCCCTTGACTACTGGGGC336    AlaSerSerGlyAlaGlyTrpGlyLeuProSerLeuAspTyrTrpGly    100105110    CAGGGAACCCTGGTCACCGTCTCC360    GlnGlyThrLeuValThrValSer    115120    (2) INFORMATION FOR SEQ ID NO:22:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 120 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    GlnValLysLeuLeuGluSerGlyGlyGlyLeuValGlnProGlyGly    151015    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerHis    202530    AlaMetSerTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal    354045    SerAspIleSerAlaSerGlyGlySerThrTyrTyrAlaAspSerVal    505560    LysGlyArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyr    65707580    LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaLeuTyrTyrCys    859095    AlaSerSerGlyAlaGlyTrpGlyLeuProSerLeuAspTyrTrpGly    100105110    GlnGlyThrLeuValThrValSer    115120    (2) INFORMATION FOR SEQ ID NO:23:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 360 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (vii) IMMEDIATE SOURCE:    (B) CLONE: SpA3-13    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..360    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    CAGGTGAAACTGCTCGAGTCTGGGGGAGGATTGGTACAGCCTGGGGGG48    GlnValLysLeuLeuGluSerGlyGlyGlyLeuValGlnProGlyGly    151015    TCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCCAT96    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerHis    202530    GCCATGAGCTGGGTCCGCCAGGCTCCAGGGAGGGGCCTGGAGTGGGTC144    AlaMetSerTrpValArgGlnAlaProGlyArgGlyLeuGluTrpVal    354045    TCAGATATTAGTGCCAGTGGTGGTAGCACATATTATGCAGACTCCGTG192    SerAspIleSerAlaSerGlyGlySerThrTyrTyrAlaAspSerVal    505560    AAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTAT240    LysGlyArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyr    65707580    TTGCAAATGAACAGCCTGAGAGCCGAAGACACGGCCTTATATTACTGT288    LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaLeuTyrTyrCys    859095    GCGTCCAACGGCGCGGGATGGGGGCTACCTTCCCTTGACTACTGGGGC336    AlaSerAsnGlyAlaGlyTrpGlyLeuProSerLeuAspTyrTrpGly    100105110    CAGGGAACCCTGGTCACCGTCTCC360    GlnGlyThrLeuValThrValSer    115120    (2) INFORMATION FOR SEQ ID NO:24:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 120 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    GlnValLysLeuLeuGluSerGlyGlyGlyLeuValGlnProGlyGly    151015    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerHis    202530    AlaMetSerTrpValArgGlnAlaProGlyArgGlyLeuGluTrpVal    354045    SerAspIleSerAlaSerGlyGlySerThrTyrTyrAlaAspSerVal    505560    LysGlyArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyr    65707580    LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaLeuTyrTyrCys    859095    AlaSerAsnGlyAlaGlyTrpGlyLeuProSerLeuAspTyrTrpGly    100105110    GlnGlyThrLeuValThrValSer    115120    (2) INFORMATION FOR SEQ ID NO:25:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 360 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (vii) IMMEDIATE SOURCE:    (B) CLONE: SpA3-15    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..360    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:    CAGGTGAAACTGCTCGAGTCTGGGGGAGGATTGGTACAGCCTGGGGGG48    GlnValLysLeuLeuGluSerGlyGlyGlyLeuValGlnProGlyGly    151015    TCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCCAT96    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerHis    202530    GCCATGAGCTGGGTCCGCCAGGCTCCAGGGAGGGGCCTGGAGTGGGTC144    AlaMetSerTrpValArgGlnAlaProGlyArgGlyLeuGluTrpVal    354045    TCAGATATTAGTGCCAGTGGTGGTAGCACATATTATGCAGACTCCGTG192    SerAspIleSerAlaSerGlyGlySerThrTyrTyrAlaAspSerVal    505560    AAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTAT240    LysGlyArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyr    65707580    TTGCAAATGAACAGCCTGAGAGCCGAAGACACGGCCTTATATTACTGT288    LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaLeuTyrTyrCys    859095    GCGTCCAACGGCGCGGGATGGGGGCTACCTTCCCTTGACTACTGGGGC336    AlaSerAsnGlyAlaGlyTrpGlyLeuProSerLeuAspTyrTrpGly    100105110    CAGGGAACCCTGGTCACCGTCTCC360    GlnGlyThrLeuValThrValSer    115120    (2) INFORMATION FOR SEQ ID NO:26:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 120 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:    GlnValLysLeuLeuGluSerGlyGlyGlyLeuValGlnProGlyGly    151015    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerHis    202530    AlaMetSerTrpValArgGlnAlaProGlyArgGlyLeuGluTrpVal    354045    SerAspIleSerAlaSerGlyGlySerThrTyrTyrAlaAspSerVal    505560    LysGlyArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyr    65707580    LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaLeuTyrTyrCys    859095    AlaSerAsnGlyAlaGlyTrpGlyLeuProSerLeuAspTyrTrpGly    100105110    GlnGlyThrLeuValThrValSer    115120    (2) INFORMATION FOR SEQ ID NO:27:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 360 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (vii) IMMEDIATE SOURCE:    (B) CLONE: SpA3-16    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..360    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:    CAGGTGAAACTGCTCGAGTCTGGGGGAGGATTGGTACAGCCTGGGGGG48    GlnValLysLeuLeuGluSerGlyGlyGlyLeuValGlnProGlyGly    151015    TCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCCAT96    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerHis    202530    GCCATGAGCTGGGTCCGCCAGGCTCCAGGGAGGGGCCTGGAGTGGGTC144    AlaMetSerTrpValArgGlnAlaProGlyArgGlyLeuGluTrpVal    354045    TCAGATATTAGTGCCAGTGGTGGTAGCACATATTATGCAGACTCCGTG192    SerAspIleSerAlaSerGlyGlySerThrTyrTyrAlaAspSerVal    505560    AAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTAT240    LysGlyArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyr    65707580    TTGCAAATGAACAGCCTGAGAGCCGAAGACACGGCCTTATATTACTGT288    LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaLeuTyrTyrCys    859095    GCGTCCAACGGCGCGGGATGGGGGCTACCTTCCCTTGACTACTGGGGC336    AlaSerAsnGlyAlaGlyTrpGlyLeuProSerLeuAspTyrTrpGly    100105110    CAGGGAACCCTGGTCACCGTCTCC360    GlnGlyThrLeuValThrValSer    115120    (2) INFORMATION FOR SEQ ID NO:28:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 120 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:    GlnValLysLeuLeuGluSerGlyGlyGlyLeuValGlnProGlyGly    151015    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerHis    202530    AlaMetSerTrpValArgGlnAlaProGlyArgGlyLeuGluTrpVal    354045    SerAspIleSerAlaSerGlyGlySerThrTyrTyrAlaAspSerVal    505560    LysGlyArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyr    65707580    LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaLeuTyrTyrCys    859095    AlaSerAsnGlyAlaGlyTrpGlyLeuProSerLeuAspTyrTrpGly    100105110    GlnGlyThrLeuValThrValSer    115120    (2) INFORMATION FOR SEQ ID NO:29:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 360 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (vii) IMMEDIATE SOURCE:    (B) CLONE: SpA3-18    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..360    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:    GTGAAACTGCTCGAGGAGTCTGGGGGAGGATTGGTACAGCCTGGGGGG48    ValLysLeuLeuGluGluSerGlyGlyGlyLeuValGlnProGlyGly    151015    TCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCCAT96    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerHis    202530    GCCATGAGCTGGGTCCGCCAGGCTCCAGGGAGGGGCCTGGAGTGGGTC144    AlaMetSerTrpValArgGlnAlaProGlyArgGlyLeuGluTrpVal    354045    TCAGATATTAGTGCCAGTGGTGGTAGCACATATTATGCAGACTCCGTG192    SerAspIleSerAlaSerGlyGlySerThrTyrTyrAlaAspSerVal    505560    AAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTAT240    LysGlyArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyr    65707580    TTGCAAATGAACAGCCTGAGAGCCGAAGACACGGCCTTATATTACTGT288    LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaLeuTyrTyrCys    859095    GCGTCCAACGGCGCGGGATGGGGGCTACCTTCCCTTGACTACTGGGGC336    AlaSerAsnGlyAlaGlyTrpGlyLeuProSerLeuAspTyrTrpGly    100105110    CAGGGAACCCTGGTCACCGTCTCC360    GlnGlyThrLeuValThrValSer    115120    (2) INFORMATION FOR SEQ ID NO:30:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 120 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:    ValLysLeuLeuGluGluSerGlyGlyGlyLeuValGlnProGlyGly    151015    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerHis    202530    AlaMetSerTrpValArgGlnAlaProGlyArgGlyLeuGluTrpVal    354045    SerAspIleSerAlaSerGlyGlySerThrTyrTyrAlaAspSerVal    505560    LysGlyArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyr    65707580    LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaLeuTyrTyrCys    859095    AlaSerAsnGlyAlaGlyTrpGlyLeuProSerLeuAspTyrTrpGly    100105110    GlnGlyThrLeuValThrValSer    115120    (2) INFORMATION FOR SEQ ID NO:31:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 360 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (vii) IMMEDIATE SOURCE:    (B) CLONE: SpA3-39    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..360    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:    GTGAAACTGCTCGAGGAGTCTGGGGGAGGATTGGTACAGCCTGGGGGG48    ValLysLeuLeuGluGluSerGlyGlyGlyLeuValGlnProGlyGly    151015    TCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCCAT96    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerHis    202530    GCCATGAGCTGGGTCCGCCAGGCTCCAGGGAGGGGCCTGGAGTGGGTC144    AlaMetSerTrpValArgGlnAlaProGlyArgGlyLeuGluTrpVal    354045    TCAGATATTAGTGCCAGTGGTGGTAGCACATATTATGCAGACTCCGTG192    SerAspIleSerAlaSerGlyGlySerThrTyrTyrAlaAspSerVal    505560    AAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTAT240    LysGlyArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyr    65707580    TTGCAAATGAACAGCCTGAGAGCCGAAGACACGGCCTTATATTACTGT288    LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaLeuTyrTyrCys    859095    GCGTCCAACGGCGCGGGATGGGGGCTACCTTCCCTTGACTACTGGGGC336    AlaSerAsnGlyAlaGlyTrpGlyLeuProSerLeuAspTyrTrpGly    100105110    CAGGGAACCCTGGTCACCGTCTCC360    GlnGlyThrLeuValThrValSer    115120    (2) INFORMATION FOR SEQ ID NO:32:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 120 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:    ValLysLeuLeuGluGluSerGlyGlyGlyLeuValGlnProGlyGly    151015    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerHis    202530    AlaMetSerTrpValArgGlnAlaProGlyArgGlyLeuGluTrpVal    354045    SerAspIleSerAlaSerGlyGlySerThrTyrTyrAlaAspSerVal    505560    LysGlyArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyr    65707580    LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaLeuTyrTyrCys    859095    AlaSerAsnGlyAlaGlyTrpGlyLeuProSerLeuAspTyrTrpGly    100105110    GlnGlyThrLeuValThrValSer    115120    (2) INFORMATION FOR SEQ ID NO:33:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 360 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (vii) IMMEDIATE SOURCE:    (B) CLONE: SpA3-37    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..360    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:    CAGGTGAAACTGCTCGAGTCTGGGGGAGGATTGGTACAGCCTGGGGGG48    GlnValLysLeuLeuGluSerGlyGlyGlyLeuValGlnProGlyGly    151015    TCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCCAT96    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerHis    202530    GCCATGAGCTGGGTCCGCCAGGCTCCAGGGAGGGGCCTGGAGTGGGTC144    AlaMetSerTrpValArgGlnAlaProGlyArgGlyLeuGluTrpVal    354045    TCAGATATTAGTGCCAGTGGTGGTAGCACATATTATGCAGACTCCGTG192    SerAspIleSerAlaSerGlyGlySerThrTyrTyrAlaAspSerVal    505560    AAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTAT240    LysGlyArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyr    65707580    TTGCAAATGAACAGCCTGAGAGCCGAAGACACGGCCTTATATTACTGT288    LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaLeuTyrTyrCys    859095    GCGTCCAACGGCGCGGGATGGGGGCTACCTTCCCTTGACTACTTGGGC336    AlaSerAsnGlyAlaGlyTrpGlyLeuProSerLeuAspTyrLeuGly    100105110    GAGGGAACCCTGGTCACCGTCTCC360    GluGlyThrLeuValThrValSer    115120    (2) INFORMATION FOR SEQ ID NO:34:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 120 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:    GlnValLysLeuLeuGluSerGlyGlyGlyLeuValGlnProGlyGly    151015    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerHis    202530    AlaMetSerTrpValArgGlnAlaProGlyArgGlyLeuGluTrpVal    354045    SerAspIleSerAlaSerGlyGlySerThrTyrTyrAlaAspSerVal    505560    LysGlyArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyr    65707580    LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaLeuTyrTyrCys    859095    AlaSerAsnGlyAlaGlyTrpGlyLeuProSerLeuAspTyrLeuGly    100105110    GluGlyThrLeuValThrValSer    115120    (2) INFORMATION FOR SEQ ID NO:35:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 351 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (vii) IMMEDIATE SOURCE:    (B) CLONE: SpA3-33    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..351    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:    GTGAAACTGCTCGAGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGTG48    ValLysLeuLeuGluGluSerGlyGlyGlyLeuValGlnProGlyVal    151015    CCCCTGAGACTCTCCTGTGAAGCCTCTGGATTCCCCTTCAGTAACTAT96    ProLeuArgLeuSerCysGluAlaSerGlyPheProPheSerAsnTyr    202530    GGCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTC144    GlyMetSerTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal    354045    TCAAGTATTAGTGGAAGTGGTGATAGTACATACTACGCCGACTCCGTG192    SerSerIleSerGlySerGlyAspSerThrTyrTyrAlaAspSerVal    505560    AAGGGCCGGTTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTAT240    LysGlyArgPheThrIleSerArgAspAsnAlaLysAsnSerLeuTyr    65707580    CTGCAAATGAACAGCCTGAGAGCCGAAGACACGGCTGTGTATTACTGT288    LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCys    859095    GCGAGAGATGCATGGGATGCATTTGATATCTGGGGCCAAGGGACAATG336    AlaArgAspAlaTrpAspAlaPheAspIleTrpGlyGlnGlyThrMet    100105110    GTCACAGTCTCCTCA351    ValThrValSerSer    115    (2) INFORMATION FOR SEQ ID NO:36:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 117 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:    ValLysLeuLeuGluGluSerGlyGlyGlyLeuValGlnProGlyVal    151015    ProLeuArgLeuSerCysGluAlaSerGlyPheProPheSerAsnTyr    202530    GlyMetSerTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal    354045    SerSerIleSerGlySerGlyAspSerThrTyrTyrAlaAspSerVal    505560    LysGlyArgPheThrIleSerArgAspAsnAlaLysAsnSerLeuTyr    65707580    LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCys    859095    AlaArgAspAlaTrpAspAlaPheAspIleTrpGlyGlnGlyThrMet    100105110    ValThrValSerSer    115    (2) INFORMATION FOR SEQ ID NO:37:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 369 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (vii) IMMEDIATE SOURCE:    (B) CLONE: SpA2-08    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..369    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:    CAGGTGAAACTGCTCGAGTCTGGGGGAACCTTGGTACAGCCGGGGGGG48    GlnValLysLeuLeuGluSerGlyGlyThrLeuValGlnProGlyGly    151015    TCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTAT96    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerTyr    202530    GCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTC144    AlaMetSerTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal    354045    TCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTG192    SerAlaIleSerGlySerGlyGlySerThrTyrTyrAlaAspSerVal    505560    AAGGGCCTATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTAT240    LysGlyLeuPheThrIleSerArgAspAsnAlaLysAsnThrLeuTyr    65707580    CTGCAGCTGAACAGTCTGAGAGCCGAGGACACGGCTGTGTATTACTGT288    LeuGlnLeuAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCys    859095    GCAAGAGCCGAGTTAGGAGGACTGATGGTTATCGTAACCCCCTCTGAG336    AlaArgAlaGluLeuGlyGlyLeuMetValIleValThrProSerGlu    100105110    TACTGGGGCCAGGGAACCCTGGTCACCGTCTCC369    TyrTrpGlyGlnGlyThrLeuValThrValSer    115120    (2) INFORMATION FOR SEQ ID NO:38:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 123 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:    GlnValLysLeuLeuGluSerGlyGlyThrLeuValGlnProGlyGly    151015    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerTyr    202530    AlaMetSerTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal    354045    SerAlaIleSerGlySerGlyGlySerThrTyrTyrAlaAspSerVal    505560    LysGlyLeuPheThrIleSerArgAspAsnAlaLysAsnThrLeuTyr    65707580    LeuGlnLeuAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCys    859095    AlaArgAlaGluLeuGlyGlyLeuMetValIleValThrProSerGlu    100105110    TyrTrpGlyGlnGlyThrLeuValThrValSer    115120    (2) INFORMATION FOR SEQ ID NO:39:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 360 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (vii) IMMEDIATE SOURCE:    (B) CLONE: SpA1-30    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..360    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:    GTGAAACTGCTCGAGCAGTCTGGGGGAGGATTGGTACAGCCTGGGGGG48    ValLysLeuLeuGluGlnSerGlyGlyGlyLeuValGlnProGlyGly    151015    TCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCCAT96    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerHis    202530    GCCATGAGCTGGGTCCGCCAGGCTCCAGGGAGGGGCCTGGAGTGGGTC144    AlaMetSerTrpValArgGlnAlaProGlyArgGlyLeuGluTrpVal    354045    TCAGATATTAGTGCCAGTGGTGGTAGCACATATTATGCAGACTCCGTG192    SerAspIleSerAlaSerGlyGlySerThrTyrTyrAlaAspSerVal    505560    AAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTAT240    LysGlyArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyr    65707580    TTGCAAATGAACAGCCTGAGAGCCGAAGACACGGCCTTATATTACTGT288    LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaLeuTyrTyrCys    859095    GCGTCCAACGGCGCGGGATGGGGGCTACCTTCCCTTGACTACTGGGGC336    AlaSerAsnGlyAlaGlyTrpGlyLeuProSerLeuAspTyrTrpGly    100105110    CAGGGAACCCTGGTCACCGTCTCC360    GlnGlyThrLeuValThrValSer    115120    (2) INFORMATION FOR SEQ ID NO:40:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 120 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:    ValLysLeuLeuGluGlnSerGlyGlyGlyLeuValGlnProGlyGly    151015    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerHis    202530    AlaMetSerTrpValArgGlnAlaProGlyArgGlyLeuGluTrpVal    354045    SerAspIleSerAlaSerGlyGlySerThrTyrTyrAlaAspSerVal    505560    LysGlyArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyr    65707580    LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaLeuTyrTyrCys    859095    AlaSerAsnGlyAlaGlyTrpGlyLeuProSerLeuAspTyrTrpGly    100105110    GlnGlyThrLeuValThrValSer    115120    (2) INFORMATION FOR SEQ ID NO:41:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 345 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (vii) IMMEDIATE SOURCE:    (B) CLONE: SpA1-29    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..345    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:    CAGGTGAAACTGCTCGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGG48    GlnValLysLeuLeuGluSerGlyGlyGlyLeuValGlnProGlyGly    151015    TCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAATTAT96    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerAsnTyr    202530    GCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTC144    AlaMetSerTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal    354045    TCAGGTATTAGTGCCAGTGGTGATACCACATACTACGCAGACTCCGTG192    SerGlyIleSerAlaSerGlyAspThrThrTyrTyrAlaAspSerVal    505560    AGGGGCCGGTTCGCCATCTCCAGAGACAATTTCAAGAACACGCTGTAT240    ArgGlyArgPheAlaIleSerArgAspAsnPheLysAsnThrLeuTyr    65707580    CTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGT288    LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCys    859095    GGTAGAGGATACAGCTACCCTGTCTGGGGGCAAGGGACCACGGTCACC336    GlyArgGlyTyrSerTyrProValTrpGlyGlnGlyThrThrValThr    100105110    GTCTCCTCA345    ValSerSer    115    (2) INFORMATION FOR SEQ ID NO:42:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 115 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:    GlnValLysLeuLeuGluSerGlyGlyGlyLeuValGlnProGlyGly    151015    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerAsnTyr    202530    AlaMetSerTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal    354045    SerGlyIleSerAlaSerGlyAspThrThrTyrTyrAlaAspSerVal    505560    ArgGlyArgPheAlaIleSerArgAspAsnPheLysAsnThrLeuTyr    65707580    LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCys    859095    GlyArgGlyTyrSerTyrProValTrpGlyGlnGlyThrThrValThr    100105110    ValSerSer    115    (2) INFORMATION FOR SEQ ID NO:43:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 372 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (vii) IMMEDIATE SOURCE:    (B) CLONE: SpA1-14    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..372    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:    CAGGTGAAACTGCTCGAGTCTGGGGGAGGCTTAGTTCAGCCTGGGGGG48    GlnValLysLeuLeuGluSerGlyGlyGlyLeuValGlnProGlyGly    151015    TCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGTTAC96    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerTyr    202530    TGGATGCACTGGGTCCGCCAAGCTCCAGGGAAGGGCCTGGTGTGGGTC144    TrpMetHisTrpValArgGlnAlaProGlyLysGlyLeuValTrpVal    354045    TCACGTATTAACACTGATGGGAGTAGAACAAGTTACGCGGACTCCGTG192    SerArgIleAsnThrAspGlySerArgThrSerTyrAlaAspSerVal    505560    AAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACCCTGTAT240    LysGlyArgPheThrIleSerArgAspAsnAlaLysAsnThrLeuTyr    65707580    CTGCAACTGAACAGTCTGAGAGCCGAGGACACGGCTGTGTATTACTGT288    LeuGlnLeuAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCys    859095    GCAAGAGCCGAATTGCGACGGCCCAATGGTTATGCTAACCCCCCTCCT336    AlaArgAlaGluLeuArgArgProAsnGlyTyrAlaAsnProProPro    100105110    GAGTACTGGGGCCAGGGAACCCTGGTCACCGTCTCC372    GluTyrTrpGlyGlnGlyThrLeuValThrValSer    115120    (2) INFORMATION FOR SEQ ID NO:44:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 124 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:    GlnValLysLeuLeuGluSerGlyGlyGlyLeuValGlnProGlyGly    151015    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerTyr    202530    TrpMetHisTrpValArgGlnAlaProGlyLysGlyLeuValTrpVal    354045    SerArgIleAsnThrAspGlySerArgThrSerTyrAlaAspSerVal    505560    LysGlyArgPheThrIleSerArgAspAsnAlaLysAsnThrLeuTyr    65707580    LeuGlnLeuAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCys    859095    AlaArgAlaGluLeuArgArgProAsnGlyTyrAlaAsnProProPro    100105110    GluTyrTrpGlyGlnGlyThrLeuValThrValSer    115120    (2) INFORMATION FOR SEQ ID NO:45:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 339 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (vii) IMMEDIATE SOURCE:    (B) CLONE: 0-19    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..339    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:    CAGGTGAAACTGCTCGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGAGG48    GlnValLysLeuLeuGluSerGlyGlyGlyLeuValGlnProGlyArg    151015    TCCCTGAGACTGTCCTGTACAGCGTCTGGATTCACCTTCAGTACCTTT96    SerLeuArgLeuSerCysThrAlaSerGlyPheThrPheSerThrPhe    202530    GCCATGAACTGGGTCCGCCAGGCTCCAGGCAAGGGCCTGGAGTGGGTC144    AlaMetAsnTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal    354045    GCAGTTGTATGGTATGATGGAACTACTAAGTACTATGCAGACTCCGTG192    AlaValValTrpTyrAspGlyThrThrLysTyrTyrAlaAspSerVal    505560    CAGGGCCGATTCACCATCTCTAGAGACAACTCCGAGAACACCCTGTAT240    GlnGlyArgPheThrIleSerArgAspAsnSerGluAsnThrLeuTyr    65707580    CTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCTGTCTATTACTGT288    LeuGlnMetAsnSerLeuArgValGluAspThrAlaValTyrTyrCys    859095    GCGAAAGGATACCAGTTGTTGCATGGGCAGACCCTGGTCACCGTCTCC336    AlaLysGlyTyrGlnLeuLeuHisGlyGlnThrLeuValThrValSer    100105110    TCA339    Ser    (2) INFORMATION FOR SEQ ID NO:46:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 113 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:    GlnValLysLeuLeuGluSerGlyGlyGlyLeuValGlnProGlyArg    151015    SerLeuArgLeuSerCysThrAlaSerGlyPheThrPheSerThrPhe    202530    AlaMetAsnTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal    354045    AlaValValTrpTyrAspGlyThrThrLysTyrTyrAlaAspSerVal    505560    GlnGlyArgPheThrIleSerArgAspAsnSerGluAsnThrLeuTyr    65707580    LeuGlnMetAsnSerLeuArgValGluAspThrAlaValTyrTyrCys    859095    AlaLysGlyTyrGlnLeuLeuHisGlyGlnThrLeuValThrValSer    100105110    Ser    (2) INFORMATION FOR SEQ ID NO:47:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 294 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (vii) IMMEDIATE SOURCE:    (B) CLONE: VH26C    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..294    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:    GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGG48    GluValGlnLeuLeuGluSerGlyGlyGlyLeuValGlnProGlyGly    151015    TCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTAT96    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerTyr    202530    GCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTC144    AlaMetSerTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal    354045    TCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGGAGACTCCGTG192    SerAlaIleSerGlySerGlyGlySerThrTyrTyrGlyAspSerVal    505560    AAGGGCCGGTTCACCATCTCCAGAGACATTTCCAAGAACACGCTGTAT240    LysGlyArgPheThrIleSerArgAspIleSerLysAsnThrLeuTyr    65707580    CTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGT288    LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCys    859095    GCGAAA294    AlaLys    (2) INFORMATION FOR SEQ ID NO:48:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 98 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:    GluValGlnLeuLeuGluSerGlyGlyGlyLeuValGlnProGlyGly    151015    SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerTyr    202530    AlaMetSerTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal    354045    SerAlaIleSerGlySerGlyGlySerThrTyrTyrGlyAspSerVal    505560    LysGlyArgPheThrIleSerArgAspIleSerLysAsnThrLeuTyr    65707580    LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCys    859095    AlaLys    (2) INFORMATION FOR SEQ ID NO:49:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 25 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (vii) IMMEDIATE SOURCE:    (B) CLONE: DomD 5'    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..25    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:    GACTCTCAAGATCTAAAAGCTGATG25    (2) INFORMATION FOR SEQ ID NO:50:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 42 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (vii) IMMEDIATE SOURCE:    (B) CLONE: DomDAS 3'    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..42    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:    TTTGTGGATCCTTCTTCTTGAGCTCCTTGGTACCTTTCGGTG42    (2) INFORMATION FOR SEQ ID NO:51:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 62 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (vii) IMMEDIATE SOURCE:    (B) CLONE: SpA Domain D VH3 Ig Binding Region    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..62    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:    AlaAspAlaGlnGlnAsnAsnPheAsnLysAspGlnGlnSerAlaPhe    151015    TyrGluIleLeuAsnMetProAsnLeuAsnGluAlaGlnArgAsnGly    202530    GluPheIleGlnSerThrLysAspAspProSerGlnSerThrAsnVal    354045    LeuGlyGluAlaLysLysLeuAsnGluSerGlnAlaProLys    505560    __________________________________________________________________________

I claim:
 1. A composition comprising a peptide and a microbialpolysaccharide antigen or glycoprotein antigen wherein the peptide is aB cell superantigen with Fab antibody binding specificity and the aminoacid sequence of SEQ. ID. No.
 51. 2. A composition according to claim 1wherein the microbial polysaccharide antigen stimulates production invertebrates of antibodies restricted to the V_(H) 3 family.
 3. A methodfor stimulating the production of variable (V) region family restrictedantibodies against one or more microbial polysaccharide antigens orglycoprotein antigens in a vertebrate host, the methodcomprising:administering a peptide wherein the peptide is a B cellsuperantigen with Fab antibody binding specificity and the amino acidsequence of SEQ. ID. No. 51 concomitantly with at least one of saidmicrobial polysaccharide antigens or glycoprotein antigens to the host,wherein said administering stimulates B cell proliferation andproduction by the host of variable region restricted antibodies withbinding specificity for the microbial polysaccharide antigens orglycoprotein antigens.